Pre-mixes of GLP-1 and basal insulin

ABSTRACT

The present invention encompasses pre-mixed formulations comprising a GLP-1 polypeptide and a basal insulin.

This is the national phase application, under 35 USC 371, forPCT/US02/21856, filed Aug. 23, 2002, which claims the priority of U.S.provisional application Nos. 60/386,061, filed Jun. 4, 2002, 60/385,266,filed May 31, 2002, and 60/315,460, filed Aug. 28, 2001.

The present invention relates to pre-mixed formulations of a glucagonlike peptide and a basal insulin. These pre-mixed formulations can beused to treat diseases such as non-insulin dependent diabetes mellitusand insulin dependent diabetes mellitus.

It has long been the goal of diabetes therapy to administer drugs thatresult in a pattern of insulin secretion that mimics the pattern ofendogenous insulin secretion in normal individuals. The dailyphysiological demand for insulin fluctuates and can be separated intotwo phases: (a) the absorptive phase requiring a pulse of insulin todispose of the meal-related blood glucose surge, and (b) the postabsorptive phase requiring a sustained delivery of insulin to regulatehepatic glucose output for maintaining optimal fasting blood glucose.

Once oral agents fail to adequately control blood glucose in type 2diabetics, it becomes extremely important to achieve near normalglycemic control and thereby minimize the complications associated withdiabetes. When oral agents fail, the only alternative is to treatpatients with insulin that must be dosed and timed with respect tomeal-related glucose excursions and hepatic glucose output duringperiods of fasting so as to effectively normalize glucose withoutcausing hypoglycemia. Control of the first phase involving disposal ofthe meal-related blood glucose surge is often the most difficult toachieve without producing side effects such as hypoglycemia. This isbecause the dose of medications must be timed such that blood insulinlevels peak when glucose levels surge after a meal. For example, ifinsulin-inducing medication is taken too long before a meal there is asubstantial risk of hypoglycemia which can result in a coma or evendeath. Furthermore, if the medication is taken too long after a mealthen blood glucose levels will remain high after a meal, which over aperiod of time can cause severe complications.

Various commercially available insulin formulations with different timeactions have been developed. However, it is often quite difficult for atype 2 patient to transition from a treatment involving oral medicationsto one involving injections of insulin that must be carefully timed withmeals to avoid complications such as hypoglycemia. Thus, there is a needfor a convenient therapy that adequately treats this intermediate stageof type 2 diabetes with a reduced risk of hypoglycemia.

Glucagon-like peptide-1 (GLP-1) shows great promise as a treatment fortype 2 diabetes especially for those patients no longer able to controlblood glucose with oral medications. GLP-1 has a variety ofphysiologically significant activities. For example, GLP-1 has beenshown to stimulate insulin release, lower glucagon secretion, inhibitgastric emptying, and enhance glucose utilization. [Nauck, M. A., etal., (1993) Diabetologia 36:741-744; Gutniak, M., et al., (1992) NewEngland J. of Med. 326:1316-1322; Nauck, M. A., et al., (1993) J. Clin.Invest. 91:301-307]. Furthermore, some animals studies suggest thatGLP-1 may actually preserve beta cells, inhibit beta cell apoptosis, andinduce beta cell proliferation. One of the most exciting observations isthat GLP-1 activity is controlled by blood glucose levels. When levelsdrop to a certain threshold level, GLP-1 is not active. Thus, there isno risk of hypoglycemia associated with treatment involving GLP-1.

However, the usefulness of a monotherapy involving GLP-1 peptides hasbeen limited by their fast clearance and short half-lives. Althoughnumerous analogs and derivatives have been developed with a longerhalf-life compared to native GLP-1(7-37)OH, the activity profile ofthese molecules is generally still not sufficient to adequately controlfasting glucose levels especially between meals and during the bedtimehours.

The present invention overcomes the problems associated with usingrelatively-short acting GLP-1 compounds to treat type 2 diabetes as wellas the hypoglycemic risk associated with insulin therapy. The presentinvention encompasses pre-mixed formulations comprising a GLP-1 compoundand a basal insulin. The GLP-1 in the mixture normalizes meal-relatedblood glucose excursions without the risk of hypoglycemia and the basalinsulin functions to control fasting blood glucose levels especiallyduring bedtime hours. In addition to providing optimal glycemic controlwith a reduced risk of hypoglycemia, a treatment regimen that employsthe pre-mixed formulations of the present invention is more convenientthan treatment with insulin alone in that doses do not need to be timedas carefully with meals because GLP-1 compounds do not causehypoglycemia.

The combination of GLP-1 and basal insulin as a pre-mixed formulationhas not been studied or even suggested. It was not understood until thepresent invention whether GLP-1 and basal insulin could be formulatedtogether such that both agents are chemically and physically stable andretain the desired time action. The molecular interactions between GLP-1and insulin could compromise the time action of either agent.Furthermore, the conditions necessary to achieve chemical and physicalstability are different for each agent when formulated alone. Until thepresent invention one skilled in the art would not appreciate that thetwo agents may be formulated together to achieve optimal glycemiccontrol in a stable, pharmaceutical formulation.

Thus, in the present invention, it was surprising that pre-mixedformulations could be prepared such that the GLP-1 compound and thebasal insulin present in the formulation produce a profile of action andphysiological response similar to that obtained when the GLP-1 and thebasal insulin compounds are injected separately.

In one form thereof, the present invention provides pre-mixedformulations comprising a GLP-1 compound and a basal insulin.

The present invention further provides a process of preparing thepre-mixed formulations, which comprises mixing a GLP-1 compound and abasal insulin in an aqueous solution such that the GLP-1 retainsinsulinotropic activity while the basal insulin retains a profile ofaction that is consistent with that produced by treatment with basalinsulin alone. Preferably, the pre-mixed formulation is prepared bymixing a stock solution of a GLP-1 compound with a basal insulin atvarious ratios. Preferably, a pharmaceutically acceptable buffer, apreservative, or an isotonicity agent may be added to the pre-mixedformulation.

The present invention further provides a method of administering aneffective amount of a pre-mixed formulation comprising a GLP-1 compoundand a basal insulin.

The present invention further provides a method of treating non-insulindependent diabetes, insulin dependent diabetes, hyperglycemia, obesity,functional dyspepsia, irritable bowel syndrome, catabolic changes aftersurgery, myocardial infarction, or stroke using the formulationsdiscussed herein.

The present invention further provides use of the formulation for thepreparation of a medicament in the treatment of non-insulin dependentdiabetes, insulin dependent diabetes, hyperglycemia, obesity,therapeutic reduction of body weight in a human subject, functionaldyspepsia, irritable bowel syndrome, catabolic changes after surgery,myocardial infarction, and stroke in a mammal.

FIG. 1 is a graphical representation of the glucose infusion ratesmeasured in dogs following initiation of a 3 hour hyperglycemic clamp at150 mg/dl and subcutaneous (SC) administration of Val⁸-GLP-1/NPH mixtureformulation (Mixture), separate Val⁸-GLP-1 solution and insulin-NPHsuspension SC administrations (Separate Sites), or insulin-NPHsuspension SC administration (NPH).

FIG. 2 is a graphical representation of the plasma insulinconcentrations (gU/mL) in dogs following initiation of a 3 hourhyperglycemic clamp at 150 mg/dl and SC administration of Val⁸-GLP-1/NPHmixture formulation (Mixture), separate Val⁸-GLP-1 solution andinsulin-NPH suspension SC administrations (Separate Sites), orinsulin-NPH suspension SC administration (NPH).

FIG. 3 is a graphical representation of the plasma C-peptideconcentrations (ng/mL) in dogs following initiation of a 3 hourhyperglycemic clamp at 150 mg/dl and SC administration of Val⁸-GLP-1/NPHmixture formulation (Mixture), separate Val⁸-GLP-1 solution andinsulin-NPH suspension SC administrations (Separate Sites), orinsulin-NPH suspension SC administration (NPH).

FIG. 4 is a graphical representation of the immunoreactive Val⁸-GLP-1(PM) in dogs following initiation of a 3 hour hyperglycemic clamp at 150mg/dl and SC administration of Val⁸-GLP-1/NPH mixture formulation(Mixture), separate Val⁸-GLP-1 solution and insulin-NPH suspension SCadministrations (Separate Sites), or insulin-NPH suspension SCadministration (NPH).

The three-letter abbreviation code for amino acids used in thisspecification conforms with the list contained in Table 3 of Annex C,Appendix 2 of the PCT Administrative Instructions and with 37 C.F.R. §1.822(d)(1)(2000).

The term “pre-mixed formulations” of the present invention refers tobiphasic or soluble formulations which comprise a GLP-1 compound and abasal insulin. As is the custom in the art) the N-terminal residue of aGLP-1 compound is represented as position 7. In the nomenclature usedherein to describe GLP-1 analogs, the substituting amino acid and itsposition is indicated prior to the parent structure. For exampleVal⁸-GLP-1(7-37)OH designates a GLP-1 analog in which the alaninenormally found at position 8 in GLP-1(7-37)OH is replaced with valine.

For purposes of the present invention, the term “GLP-1 compound” as usedherein refers to polypeptides that include naturally occurring truncatedGLP-1 polypeptides (GLP-1(7-37)OH and GLP-1(7-36)NH₂), GLP-1 fragments,GLP-1 analogs, and derivatives thereof. For purposes of the presentinvention, GLP-1 compounds also include Exendin-3 and Exendin-4, andanalogs and derivatives thereof. GLP-1 compounds of the presentinvention have the ability to bind to the GLP-1 receptor and initiate asignal transduction pathway resulting in insulinotropic activity.Examples of GLP-1 compounds appropriate for use in the present inventionare discussed more extensively below.

The term “insulinotropic activity” refers to the ability to stimulateinsulin secretion in response to elevated glucose levels, therebycausing glucose uptake by cells and an associated decrease in plasmaglucose levels. Insulinotropic activity can be assessed by methods knownin the art, including using in vivo experiments and in vitro assays thatmeasure GLP-1 receptor binding activity or receptor activation, e.g.,assays employing pancreatic islet cells or insulinoma cells, asdescribed in EP 619,322 to Gelfand, et al. (described in Example 1), andU.S. Pat. No. 5,120,712, respectively. Insulinotropic activity isroutinely measured in humans by measuring insulin levels or C-peptidelevels. A GLP-1 compound has insulinotropic activity if islet cellssecrete insulin levels in the presence of the GLP-1 compound abovebackground levels.

GLP-1 compounds can exist in at least two different forms. The firstform is physiologically active and dissolves readily in aqueous solutionat physiological pH (7.4). In contrast, the second form has little or noinsulinotropic activity and is substantially insoluble in water at pH7.4. Thus, the GLP-1 compound should be formulated under conditions thatreduce the propensity of the GLP-1 compound to aggregate and generate aninsoluble, inactive complex. The propensity of a particular GLP-1compound under a given set of formulation conditions may be determinedby measuring turbidity at 350 nm as described in Example 8.

Preferably the pre-mixed formulations encompassed by the presentinvention are comprised of a GLP-1 compound with insulinotropic activitythat is equal to or greater than GLP-1 (7-37)OH. It is even morepreferable that the GLP-1 compound have greater insulinotropic activitythan GLP-1(7-37)OH.

The term “basal insulin” used in the present invention refers to aninsulin analog having basal activity or a formulation of an insulin orinsulin analog that has basal activity. Generally, basal insulins arerecognized in the art and exhibit protracted time action greater than 8hours in standard models of diabetes. Preferably, the basal insulin hasa basal activity of about 24 hours. Preferably, the basal insulin has abasal activity of 8 to 14 hours. Preferably, the basal insulin has abasal activity similar to that observed for commercial formulations ofNPH, NPL, PZI, Ultralente, or insulin glargine.

The term “biphasic formulation” used in the present invention refers toa basal insulin that is substantially insoluble and a GLP-1 compoundthat is substantially soluble in the pre-mixed formulation. The basalinsulin is substantially insoluble if after centrifugation of themixture little or no insulin is detected in the supernatant. Preferably,the insoluble insulin in the formulation remains insoluble for anextended period of time under the conditions of storage. The protractedtime action of these basal insulins is due in part to the slow rate ofabsorption and dissociation of a hexamer of insulin to the activeinsulin monomers after injection.

The term “solution formulation” used in the present invention refers toa basal insulin that is substantially soluble and a GLP-1 compound thatis substantially soluble in the pre-mixed formulation. Examples ofsoluble basal insulins include insulins wherein the isoelectric point(pI) is shifted and acylated insulins. Insulin glargine (Lantus®) is oneexample of a pI shifted insulin. By addition to and or substitution ofbasic amino acids in regular human insulin, the pI is shifted from about5.5 to a more neutral pH. For example, addition of two arginines at theC-terminal end of the B chain results in a shift of the pI to about 7.This pI shifted insulin analog would be soluble at an acidic or basicpH. Alternatively, the pI can be shifted more acidic by addition to andor substitution of acidic amino acids in regular human insulin. This pIshifted insulin analog would be soluble at a basic pH. Then, uponinjection of either solution, the pH will adjust to a physiological pHsufficiently close to the pI of the insulin analog, the net charge willbe zero, and the result will be precipitation of the insulin. Theprecipitated insulin then slowly dissolves and is absorbed into theblood over a period of time to achieve the desired basal activity.

Acylated insulins are generally described as an insulin or insulinanalog having lipophilic substituents on the insulin. The lipophilicsubstituents interact with proteins in the blood such as albumin. Theresult is that the insulin is preserved for a longer period of timewhile circulating in the blood. In addition, the lipophilic substituentsmay provide increased stability by protecting the insulin fromdegradative enzymes. Further, the lipophilic substituents may delayabsorption of the insulin into the blood from the injection site.

The ratio of GLP-1 compound to basal insulin is such that afteradministration of the formulation, the plasma levels are maintainedwithin the efficacious range. Preferably, serum levels of a GLP-1compound that has insulinotropic activity within 2-fold that ofGLP-1(7-37)OH is maintained between about 30 picomoles/liter and about200 picomoles/liter. Optimum serum levels will be higher for GLP-1compounds that are less active than GLP-1(7-37)OH or lower for GLP-1compounds that are more active than GLP-1(7-37)OH. In general, themixture will be formulated such that about 0.1 to about 5 mg of GLP-1compound will be administered per day. Preferably, the GLP-1 compoundwill be administered in the range of 0.1 to 2 mg/day. More preferably,the GLP-1 compound will be administered in the range of 0.5 to 2 mg/day.The concentration of the GLP-1 compound may be adjusted upwards ordownwards depending on the activity of the GLP-1 compound selected. Theconcentration of the GLP-1 compound in the premixed formulation ingeneral is in the range of 0.1 to 20 mg/ml. Preferably, concentration ofthe GLP-1 compound in the premixed formulation is in the range of 0.1 to10 mg/ml. More preferably, the concentration of the GLP-1 compound is inthe range of 0.1 to 5 mg/ml.

In general, the mixture will be formulated such that about 0.01 to about1 U/kg of basal insulin will be administered per day. Preferably, thebasal insulin will be administered in the range of 0.05 to 0.5 U/kg/day.More preferably, the basal insulin will be administered in the range of0.05 to 0.3 U/kg/day.

The various pre-mixed formulations comprising a GLP-1 compound and abasal insulin of the present invention may optionally encompass apharmaceutically acceptable buffer. However, the selection,concentration, and pH of the buffer shall be such that the GLP-1compound remains substantially soluble in the formulation and retainsinsulinotropic activity and the basal insulin retains a protractedaction profile. Examples of pharmaceutically acceptable buffers includephosphate buffers like dibasic sodium phosphate, TRIS, acetate, such assodium acetate, citrate, such as sodium citrate, sodium tartarate, basicamino acids such as histidine, lysine or arginine, or neutral aminoacids such as glycine and glycyl-glycine. Other pharmaceuticallyacceptable buffers are known in the art. Preferably, the buffer isselected from the group consisting of acetate, phosphate and TRIS. Theskilled artisan will recognize that the selection of the buffer isdependent upon the desired pH and the pKa of the buffer. Thus, in thecase where the desired pH is in the physiological range, the bufferswith a pKa in that range are desired. Preferably the buffer is phosphateand TRIS when the pH is in the physiological range. Where the desired pHis in the basic range, a preferred buffer is TRIS and where the desiredpH is in the acidic range, a preferred buffer is acetate. Preferably,the concentration of a buffer is between about 1 mM and 30 mM. Even morepreferably, the concentration is between about 4 mM and 14 mM

The pH of the pre-mixed formulation is adjusted to provide acceptablestability, to maintain the solubility and insulinotropic activity of theGLP-1 compound and the protracted action profile of the basal insulinand be acceptable for parenteral administration. When the basal insulinis insoluble, the pH of the pre-mixed formulation is preferably adjustedto between about 7.0 and about 8.5, more preferably the pH is betweenabout 7.4 and 8.0, even more preferably the pH is between about 7.4 and7.8. Most preferably, the pH is between about 7.6 and 7.8, mostpreferably 7.8.

However, when the basal insulin is a pI shifted insulin analog, the pHis adjusted to maintain solubility of both the GLP-1 compound and thebasal insulin analog in an aqueous medium. For example, human insulinhas a pI of about 5.5. When the pI is shifted up to about 7 as a resultof the addition of basic amino acids, the pH is adjusted to slightlyacidic or slightly basic pH to maintain solubility. The pH can beadjusted to less than about 6, less than about 5, less than about 4. ThepH can also be adjusted to greater than about 8, greater than about 9,greater than about 10. When the pI shifted insulin analog is insulinglargine, the pH is adjusted to about 4. Alternatively, when the pIshifted insulin analog is insulin glargine, the pH can be adjusted toabout 8.

The pH is also dependent upon the GLP-1 compounds used in the premixedformulations. In general, the pH of the GLP-1 compounds is between about4 and about 10. The pH is adjusted to slightly acidic or slightly basicpH to maintain solubility dependent on the pI of the GLP-1 compound.When the pI of the GLP-1 compound is about 7 then the pH can be adjustedto less than about 6, less than about 5, about 4. The pH can also beadjusted to greater than about 8, greater than about 9, about 10. Forexample, GLP-1 compounds with glutamic acid at position 22 can beformulated at an acidic pH and still remain soluble. Preferably, the pHof GLP-1 compounds with glutamic acid at position 22 is between about 4and 6. More preferably the pH is about 4. Other GLP-1 compounds with aneutral amino acid at position 22 can be formulated at physiological orhigher pH and still remain soluble. Preferably, the pH of GLP-1compounds with a neutral amino acid at position 22 is between about 7and 10. More preferably the pH is between about 7 and 8.5. Exendin-3 andExendin-4 can be formulated at an acidic pH and still remain soluble.Preferably, the pH of Exendin-3 or Exendin-4 is between about 4 and 6.More preferably the pH is about 4.

The pre-mixed formulations of the present invention may optionallyencompass a preservative. However, the selection and concentration ofthe preservative shall be such that the GLP-1 compound remainssubstantially soluble in the formulation and retains insulinotropicactivity and the basal insulin retains a protracted action profile.Preservative refers to a compound that is added to a pharmaceuticalformulation to act as an anti-microbial agent. A parenteral formulationmust meet guidelines for preservative effectiveness to be a commerciallyviable multi-use product. Among preservatives known in the art as beingeffective and acceptable in parenteral formulations are phenolicpreservatives, alkylparabens, benzyl alcohol, chlorobutanol, resorcinol,and other similar preservatives, and various mixtures thereof. Examplesof phenolic derivatives include cresols and phenol or a mixture ofcresols and phenol. Examples of cresols include meta-cresol,ortho-cresol, para-cresol, chloro-cresol, or mixtures thereof.Alkylparaben refers to a C₁ to C₄ alkyl paraben, or mixtures thereof.Examples of alkylparabens include methylparaben, ethylparaben,propylparaben, or butylparaben. The concentration of the preservative isknown to one skilled in the art. The concentrations must be sufficientto maintain preservative effectiveness by retarding microbial growth.Preferably, the preservative is a phenol derivative. More preferably thepreservative is cresol, phenol, or a mixture of cresol and phenol. Evenmore preferably the preservative is meta-cresol, phenol, or a mixture ofmeta-cresol and phenol.

For biphasic formulations, the preferred preservative is a mixture ofmeta-cresol and phenol. In general, the concentration of meta-cresol isbetween about 0.1 to about 4.0 mg/mL. The preferred concentration ofmeta-cresol is about 1.6 mg/mL. In general, the concentration of phenolis between about 0.1 to about 2.0 mg/mL. The preferred concentration ofphenol is about 0.65 mg/mL.

For soluble formulations, the preferred preservative is meta-cresol orphenol. In general, the concentration of meta-cresol is between about2.0 to about 8.0 mg/mL, about 2.5 mg/mL to about 4.5 mg/mL, and about2.0 mg/mL to about 4.0 mg/mL. A most preferred concentration ofpreservative in the final mixture is about 2.7 mg/mL. In anotherembodiment, the concentration of phenol is between about 2.0 to about10.0 mg/mL, and about 4.0 to about 8.0 mg/mL. A most preferredconcentration of preservative in the final mixture is about 5.0 mg/mL.

In general, insulins are converted to a hexamer complex by dissolvingthe insulin in a diluent containing the preservative in suitablequantities at a pH of about 7 to about 8 and then adding zinc. However,the selection and amount of preservative and zinc shall be such that theGLP-1 compound remains substantially soluble in the formulation andretains insulinotropic activity and the basal insulin retains aprotracted action profile. Zinc is preferably added as a zinc salt, suchas, without limitation, zinc acetate, zinc bromide, zinc chloride, zincfluoride, zinc iodide, and zinc sulfate. The skilled artisan willrecognize that there are may other zinc salts which also might be usedto make the insulin analog complexes that are part of the presentinvention. Preferably, the zinc salts are zinc acetate, zinc oxide, orzinc chloride.

In general, the hexamer complex consists of two zinc ions per hexamer ofhuman insulin analog, and at least three molecules of a phenolicpreservative selected from the group consisting of chlorocresol,m-cresol, phenol, and mixtures thereof.

The pre-mixed formulations of the present invention may optionallyencompass an isotonicity agent. However, the selection and concentrationof the isotonicity agent shall be such that the GLP-1 compound remainssubstantially soluble in the formulation and retains insulinotropicactivity and the basal insulin retains a protracted action profile.Isotonicity agents refer to compounds that are tolerated physiologicallyand impart a suitable tonicity to the formulation to prevent the netflow of water across cell membranes. Examples of such compounds includeglycerin (or glycerol), salts, e.g., NaCl, and sugars, e.g., dextrose,mannitol, and sucrose. These compounds are commonly used for suchpurposes at known concentrations. One or more isotonicity agents may beadded to adjust the ionic strength or tonicity.

For biphasic formulations, a preferred isotonicity agent is glycerin.The concentration of glycerin is preferably between about 12 mg/mL and25 mg/ml, preferably between about 12 mg/mL and 20 mg/ml, and morepreferred is about 16 mg/ml.

For soluble formulations, the preferred isotonicity agent is NaCl. Theconcentration of NaCl is preferably between about 10 mM and 200 mM, morepreferred is between about 50 mM and 150 mM, and most preferred is about100 mM. In another 2.5 embodiment, the preferred isotonicity agent ismannitol. The concentration of the mannitol is preferably between about1% (weight (w)/volume (v)) and 10% (w/v), and more preferred is betweenabout 2% (w/v) and 8% (w/v). In another embodiment, the preferredisotonicity agent is glycerin. The concentration of the glycerin ispreferably between about 12 mg/mL and 25 mg/ml, preferably between about12 mg/mL and 20 mg/ml, and more preferred is about 17 mg/ml.

Soluble formulations of the present invention may optionally encompass asolubility enhancer. However, the selection and concentration of thesolubility enhancer shall be such that the GLP-1 compound remainssubstantially soluble in the formulation and retains insulinotropicactivity and the basal insulin remains substantially soluble in theformulation and retains a protracted action profile. Solubilityenhancers provide stability to the basal insulin and the GLP-1 compound,such that the basal insulin and the GLP-1 compound remain soluble for anextended period of time under the conditions of storage. Preferably thesolubility enhancer is nicotinamide. In general, the concentration ofnicotinamide is between 0.01 and 2 molar. Other preferred ranges ofnicotinamide concentration are: between 0.05 molar and 1.5 molar;between 0.1 molar and 1.0 molar; between 0.1 molar and 0.5 molar;between 0.5 molar and 1.0 molar; and between 0.15 molar and 0.25 molar.

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20® (polyoxyethylene (20) sorbitan monolaurate), Tween 40®(polyoxyethylene (20) sorbitan monopalmitate), Tween 80®(polyoxyethylene (20) sorbitan monooleate), Pluronic F68®(polyoxyethylene polyoxypropylene block copolymers), and PEG(polyethylene glycol) may optionally be added to the formulation.Although these additives are not necessarily required, they may beuseful if the formulations will contact plastic materials.

Preferably, when injected, the premixed formulations of the presentinvention result in a glucose profile that is the same or better thanthat obtained when the GLP-1 compound and basal insulin are administeredseparately.

The pre-mixed formulations of the present invention are suitable totreat diseases or conditions wherein the physiological effects ofadministering GLP-1 or insulin improves the disease or condition.

Included are subjects with non-insulin dependent diabetes, insulindependent diabetes, stroke (see WO 00/16797 by Efendic), myocardialinfarction (see WO 98/08531 by Efendic), obesity (see WO 98/19698 byEfendic), catabolic changes after surgery (see U.S. Pat. No. 6,006,753to Efendic), functional dyspepsia and irritable bowel syndrome (see WO99/64060 by Efendic). Also included are subjects requiring prophylactictreatment with a GLP-1 compound, e.g., subjects at risk for developingnon-insulin dependent diabetes (see WO 00/07617). Additional subjectsinclude those with impaired glucose tolerance or impaired fastingglucose, subjects whose body weight is about 25% above normal bodyweight for the subject's height and body build, subjects with a partialpancreatectomy, subjects having one or more parents with non-insulindependent diabetes, subjects who have had gestational diabetes andsubjects who have had acute or chronic pancreatitis are at risk fordeveloping non-insulin dependent diabetes.

The pre-mixed formulations of the present invention can be used tonormalize blood glucose levels, prevent pancreatic β-cell deterioration,induce β-cell proliferation, stimulate insulin gene transcription,up-regulate IDX-1/PDX-1 or other growth factors, improve β-cellfunction, activate dormant β-cells, differentiate cells into β-cells,stimulate β-cell replication, inhibit β-cell apoptosis, regulate bodyweight, and induce weight loss.

The premixed formulations described herein can be used to treat subjectswith a wide variety of diseases and conditions. The GLP-1 compoundsencompassed in the premixed formulations of the present invention exerttheir biological effects by acting at a receptor referred to as the“GLP-1 receptor” (see Thorens, PNAS 89, 8641-8645 (1992)).

Subjects with diseases and/or conditions that respond favorably to GLP-1receptor stimulation or to the administration of GLP-1 compounds cantherefore be treated with the GLP-1 compounds of the present invention.These subjects are said to “be in need of treatment with GLP-1compounds” or “in need of GLP-1 receptor stimulation.”

The following provides an even more detailed discussion of the GLP-1compounds and basal insulins useful in the premixed formulations of thepresent invention.

Representative examples of GLP-1 compounds that can be used in thepre-mixed formulations of the present invention include those known inthe art such as the analogs disclosed in U.S. Pat. Nos. 5,118,666,5,120,712, 5,512,549, 6,191,102, 5,977,071, 5,545,618, 5,705,483,6,133,235, and in Adelhorst, et al., (1994) J. Biol. Chem. 269:6275.

The two naturally occurring truncated GLP-1 polypeptides are representedin formula I (SEQ ID NO: 1):

SEQ ID NO: 1 7   8   9   10  11  12  13  14  15  16  17His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-29  30  31  32  33  34  35  36  37 Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Xaa

-   -   wherein:    -   Xaa at position 37 is Gly, or —NH₂.

The term “dipeptidyl peptidase IV (DPP IV) resistant” refers to GLP-1molecules that have extended metabolic stability and improved biologicalactivity because they are resistant to the endogenous enzyme, DPP IV.For example, DPP IV resistance can be determined using the methoddescribed in Example 2. A GLP-1 molecule is DPP IV resistant if in thepresence of DPP IV the GLP-1 molecule has extended metabolic stabilityabove that of native GLP-1. DPP IV resistant GLP-1 molecules can have anamino acid change at the DPP IV recognition site (position 8), or DPP IVresistant peptides can have an attached group that restricts theaccessibility of DPP IV to the recognition site, or both.

A “GLP-1 fragment” is a polypeptide obtained after truncation of one ormore amino acids from the N-terminus and/or C-terminus of GLP-1(7-37)OHor an analog or derivative thereof. The nomenclature used to describeGLP-1(7-37)OH is also applicable to GLP-1 fragments. For example,GLP-1(9-36)OH denotes a GLP-1 fragment obtained by truncating two aminoacids from the N-terminus and one amino acid from the C-terminus. Theamino acids in the fragment are denoted by the same number as thecorresponding amino acid in GLP-1(7-37)OH. For example, the N-terminalglutamic acid in GLP-1(9-36)OH is at position 9; position 12 is occupiedby phenylalanine; and position 22 is occupied by glycine, as inGLP-1(7-37)OH. For GLP-1(7-36)OH, the Glycine at position 37 ofGLP-1(7-37)OH is deleted.

A “GLP-1 analog” has sufficient homology to GLP-1(7-37)OH or a fragmentof GLP-1(7-37)OH such that the analog has insulinotropic activity.Preferably, a GLP-1 analog has the amino acid sequence of GLP-1(7-37)OHor a fragment thereof, modified so that from one, two, three, four orfive amino acids differ from the amino acid in corresponding position ofGLP-1(7-37)OH or a fragment of GLP-1(7-37)OH. In the nomenclature usedherein to designate GLP-1 compounds, the substituting amino acid and itsposition is indicated prior to the parent structure. For example,Glu²²-GLP-1(7-37)OH designates a GLP-1 compound in which the glycinenormally found at position 22 of GLP-1(7-37)OH has been replaced withglutamic acid; Val⁸-Glu²²-GLP-1(7-37)OH designates a GLP-1 compound inwhich alanine normally found at position 8 and glycine normally found atposition 22 of GLP-1(7-37)OH have been replaced with valine and glutamicacid, respectively.

Other GLP-1 compounds of the present invention include additions of oneor more amino acids to the N-terminus and/or C-terminus of GLP-1.Preferably from one to six amino acids are added to the N-terminusand/or from one to eight amino acids are added to the C-terminus ofGLP-1. It is preferred that GLP-1 analogs of this type have up to aboutthirty-nine amino acids. The amino acids in the extended GLP-1 analogsare denoted by the same number as the corresponding amino acid inGLP-1(7-37)OH. For example, the N-terminal amino acid of a GLP-1 analogobtained by adding two amino acids to the N-terminus of GLP-1(7-37)OH isat position 5; and the C-terminal amino acid of a GLP-1 compoundobtained by adding one amino acid to the C-terminus of GLP-1(7-37)OH isat position 38. Amino acids 1-6 of an extended GLP-1 analog arepreferably the same as or a conservative substitution of the amino acidat the corresponding position of GLP-1(1-37)OH. Amino acids 38-45 of anextended GLP-1 compound are preferably the same as or a conservativesubstitution of the amino acid at the corresponding position ofExendin-4.

A conservative substitution is the replacement of an amino acid withanother amino acid that has the same net electronic charge andapproximately the same size and shape. Amino acids with aliphatic orsubstituted aliphatic amino acid side chains have approximately the samesize when the total number of carbon and heteroatoms in their sidechains differs by no more than about four. They have approximately thesame shape when the number of branches in the their side chains differsby no more than one. Amino acids with phenyl or substituted phenylgroups in their side chains are considered to have about the same sizeand shape. Preferably, a GLP-1 compound has the amino acid sequence ofSEQ ID NO. 1 or is modified so that from one, two, three, four or fiveamino acids differ from SEQ ID NO: 1.

The amino acid sequence of Exendin-3 and Exendin-4 are represented informula II (SEQ ID NO:2):

SEQ ID NO: 2 7   8   9   10  11  12  13  14  15  16  17His-Xaa-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-18  19  20  21  22  23  24  25  26  27  28Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-29  30  31  32  33  34  35  36  37  38  39Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser- 40  41  42  43  44  45Gly-Ala-Pro-Pro-Pro-Ser

-   -   wherein:    -   Xaa at position 8 is Ser or Gly;    -   Xaa at position 9 is Asp or Glu; and    -   Ser at position 45 is Ser or Ser-NH₂.        Exendin-3 has Ser at position 8 and Asp at position 9. Exendin-4        has Gly at position 8 and Glu at position 9. Other GLP-1        compounds of the present invention include Exendin-3 and        Exendin-4 agonists as described in WO99/07404, WO99/25727,        WO99/25728, WO99/43708, WO00/66629, and US2001/0047084A1 and are        herein incorporated by reference.

A preferred group of GLP-1 analogs are represented in formula III (SEQID NO:3):

SEQ ID NO: 3 7   8   9   10  11  12  13  14  15  16  17Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Xaa-Asp-Xaa-Xaa-18  19  20  21  22  23  24  25  26  27  28Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-29  30  31  32  33  34  35  36  37  38  39Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- 40  41  42  43  44  45Xaa-Xaa-Xaa-Xaa-Xaa-Xaawherein:

-   -   Xaa at position 7 is: L-histidine, D-histidine,        desamino-histidine, 2-amino-histidine, βhydroxy-histidine,        homohistidine, α-fluoromethyl-histidine or α-methyl-histidine;    -   Xaa at position 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys;    -   Xaa at position 9 is Glu, Asp, Lys, Thr, Ser, Arg, Trp, Phe,        Tyr, or His;    -   Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu,        Asp, Arg, His, or Lys;    -   Xaa at position 12 is His, Trp, Phe, or Tyr    -   Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,        Asp, or Lys;    -   Xaa at position 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr,        Glu, Asp, Trp, His, Phe, or Lys;    -   Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,        Asp, or Lys;    -   Xaa at position 18 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu,        Asp, His, Pro, Arg, or Lys;    -   Xaa at position 19 is Tyr, Phe, Trp, Glu, Asp, Gly, Gln, Asn,        Arg, Cys, or Lys;    -   Xaa at position 20 is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu,        Asp, Met, or Lys;    -   Xaa at position 21 is Glu, Asp, or Lys;    -   Xaa at position 22 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys;    -   Xaa at position 23 is Gln, Asn, Arg, Glu, Asp, His, or Lys;    -   Xaa at position 24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg,        Glu, Asp, or Lys;    -   Xaa at position 25 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,        Asp, or Lys;    -   Xaa at position 26 is Lys, Arg, Gln, Glu, Asp, Trp, Tyr, Phe, or        His;    -   Xaa at position 27 is Glu, Asp, Ala, His, Phe, Tyr, Trp, Arg,        Leu, or Lys;    -   Xaa at position 30 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu,        Asp, His, or Lys;    -   Xaa at position 31 is Trp, Phe, Tyr, Glu, Asp, Ser, Thr, Arg, or        Lys;    -   Xaa at position 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu,        Asp, or Lys;    -   Xaa at position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu,        Asp, Arg, or Lys;    -   Xaa at position 34 is Lys, Arg, Glu, Asp, Asn, or His;    -   Xaa at position 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,        Asp, Arg, Trp, Tyr, Phe, Pro, His, or Lys;    -   Xaa at position 36 is Arg, Lys, Glu, Asp, Thr, Ser, Trp, Tyr,        Phe, Gly, or His;    -   Xaa at position 37 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu,        Asp, His, Lys, Arg, Trp, Tyr, Phe, Pro, Pro-NH₂ or is deleted;    -   Xaa at position 38 is Arg, Lys, Glu, Asp, Ser, or His, or is        deleted;    -   Xaa at position 39 is Arg, Lys, Glu, Asp, Ser, or His, or is        deleted;    -   Xaa at position 40 is Asp, Glu, Gly, or Lys, or is deleted;    -   Xaa at position 41 is Phe, Trp, Tyr, Glu, Asp, Ala, or Lys, or        is deleted;    -   Xaa at position 42 is Ser, Pro, Lys, Glu, or Asp, or is deleted;    -   Xaa at position 43 is Ser, Glu, Asp, Pro, or Lys, or is deleted;    -   Xaa at position 44 is Gly, Glu, Asp, Pro, or Lys, or is deleted;        and    -   Xaa at position 45 is Ala, Val, Glu, Asp, Ser, or Lys, or        Ala-NH₂, Val-NH₂, Glu-NH₂, Asp-NH₂, Ser-NH₂, or Lys-NH₂, or is        deleted, or a C-1-6-ester, or amide, or C-1-6-alkylamide, or        C-1-6-dialkylamide thereof; provided that when the amino acid at        position 37, 38, 39, 40, 41, 42, 43, or 44 is deleted, then each        amino acid downstream of that amino acid is also deleted.

A preferred group of GLP-1 analogs are:

HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRG or G-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIDGGPSSGRPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRGSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAXEFIAWLVKGRPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLVKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGDPPPS or S-NH2NVEGTFTSDVSSYLEEQAAKEFIAWLIKGGGSSGDPPPS or S-NH2RVEGTFTSDVSSYLEEQAAKEFIAWLIKGPGSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGSPSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDAPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPAPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDPPAS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGDAAAS or S-NH2EVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPSSGAPPPH or H-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLIKGGPSSGDPPPS or S-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEGQAAKEFIAWLIKGGPHSSGAPPPS or S-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLVKGRGSSGAPPPS or S-NH2HVEGTFTSDVSSYLEGQAAKEFIAWLVKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRGSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGRGSSGAPPPS or S-NH2HVEGTFTSDWSSYLEEQAAKEFIAWLIKGRGSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGRGHSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGRGHSSGAPPPS or S-NH2HVEGTFTSDWSSYLEEQAAKEFIAWLIKGGPHSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEEQAAKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDVSWYLEGQAVKEFIAWLIKGGPHSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEEQAVKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDWSSYLEEQAVKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDWSSYLEEQAVKEFIAWLIKGGPHSSGAPPPS or S-NH2HVEGTFTSDWSKYLEEQAVKEFIAWLIKGGPSSGAPPPSH or H-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPRG or G-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLIKGGPSSGAPPPRG or G-NH₂HVEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPS or S-NH₂HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGRPPPS or S-NH₂HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGRPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGKPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVDGGPSSGRG or G-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLVKGGPSWGAPPPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPGPS or S-NH2HVEGTFTSDVSSYLEEQAAKEFIAWLIKGGPSSGAPPPGPSGPS or S-NH2HVEGTFTSDVSSYLEEQAVKEFIAWLVKGGPSSGAPPPS or S-NH2

Another preferred group of GLP-1 analogs is represented in formula IV(SEQ ID NO:4):

7   8   9   10  11  12  13  14  15  16  17His-Xaa-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Xaa-Xaa-Ala-Ala-Lys-Xaa-Phe-29  30  31  32  33  34  35  36  37 Ile-Xaa-Trp-Leu-Val-Lys-Gly-Arg-Rwherein:

-   -   Xaa at position 8 is Gly, Ala, Val, Leu, Ile, Ser, or Thr;    -   Xaa at position 22 is Asp, Glu, Gln, Asn, Lys, Arg, Cys, or        Cysteic Acid;    -   Xaa at position 23 is His, Asp, Lys, Glu, or Gln;    -   Xaa at position 27 is Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys;    -   Xaa at position 30 is Glu, Asp, Ser, or His;    -   R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, —NH₂.

It is also preferable that the GLP-1 compounds of the present inventionhave other combinations of substituted amino acids. The presentinvention encompasses a GLP-1 compound comprising the amino acidsequence of formula V (SEQ ID NO:5)

Xaa₇-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Xaa₁₉-Xaa₂₀-Glu-Xaa₂₂-Gln-Ala-Xaa₂₅-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Lys-Gly-Arg- Xaa₃₇ Formula V (SEQ IDNO: 5)wherein:

-   -   Xaa₇ is: L-histidine, D-histidine, desamino-histidine,        2-amino-histidine, β-hydroxy-histidine, homohistidine,        α-fluoromethyl-histidine, or α-methyl-histidine;    -   Xaa₈ is: Ala, Gly, Val, Leu, Ile, Ser, or Thr;    -   Xaa₁₂ is: Phe, Tip, or Tyr;    -   Xaa₁₆ is: Val, Trp, Ile, Leu, Phe, or Tyr;    -   Xaa₁₈ is: Ser, Trp, Tyr, Phe, Lys, Ile, Leu, Val;    -   Xaa₁₉ is: Tyr, Trp, or Phe;    -   Xaa₂₀ is: Leu, Phe, Tyr, or Trp;    -   Xaa₂₂ is: Gly, Glu, Asp, Lys;    -   Xaa₂₅ is: Ala, Val, Ile, or Leu;    -   Xaa₂₇ is: Glu, Ile, or Ala;    -   Xaa₃₀ is: Ala or Glu    -   Xaa₃₃ is: Val, or Ile; and    -   Xaa₃₇ is: Gly, His, NH₂, or is absent.

The present invention also encompasses a GLP-1 compound comprising theamino acid sequence of formula VI (SEQ ID NO:6)

Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Leu-Glu-Xaa₂₂-Gln-Ala-Xaa₂₅-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Xaa₃₃-Lys-Gly-Arg-Xaa₃₇ Formula VI (SEQ ID NO: 6)wherein:

-   -   Xaa₇ is: L-histidine, D-histidine, desamino-histidine,        2-amino-histidine, β-hydroxy-histidine, homohistidine,        α-fluoromethyl-histidine, or α-methyl-histidine;    -   Xaa₈ is: Gly, Ala, Val, Leu, Ile, Ser, or Thr;    -   Xaa₁₆ is: Val, Phe, Tyr, or Trp;    -   Xaa₁₈ is: Ser, Tyr, Trp, Phe, Lys, Ile, Leu, or Val;    -   Xaa₂₂ is: Gly, Glu, Asp, or Lys;    -   Xaa₂₅ is: Ala, Val, Ile, or Leu;    -   Xaa₃₃ is: Val or Ile; and    -   Xaa₃₇ is: Gly, NH₂, or is absent.

It is preferred that the backbone of the GLP-1 analogs of formula I, II,III, IV, V, and VI comprise an amino acid other than alanine at position8 (position 8 analogs). Preferred amino acids at position 8 are glycine,valine, leucine, isoleucine, serine, threonine, or methionine and morepreferably are valine or glycine. The backbone may also includeL-histidine, D-histidine, or modified forms of histidine such asdesamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, α-fluoromethyl-histidine, or α-methyl-histidine atposition 7. It is preferable that these position 8 analogs contain oneor more additional changes at positions 12, 16, 18, 19, 20, 22, 25, 27,30, 33, and 37 compared to the corresponding amino acid of nativeGLP-1(7-37)OH. It is more preferable that these position 8 analogscontain one or more additional changes at positions 16, 18, 22, 25 and33 compared to the corresponding amino acid of native GLP-1(7-37)OH.

Even more preferred are GLP-1 analogs of formula I, II, III, IV, V, andVI wherein not more than 6 amino acids differ from the correspondingamino acid in native GLP-1(7-37)OH, GLP-1(7-36)NH₂, or Exendin-4. Mostpreferred are GLP-1 analogs of formula I, II, III, and IV whereinbetween 1 and 5 amino acids differ from the corresponding amino acid innative GLP-1(7-37)OH, GLP-1(7-36)NH₂, or Exendin-4.

Other preferred GLP-1 analogs have the sequence of GLP-1(7-37)OH exceptthat the amino acid at position 8 is preferably glycine, valine,leucine, isoleucine, serine, threonine, or methionine and morepreferably valine or glycine and position 22 is glutamic acid, lysine,aspartic acid, or arginine and more preferably glutamic acid or lysine.

Other preferred GLP-1 analogs have the sequence of GLP-1(7-37)OH exceptthat the amino acid at position 8 is preferably glycine, valine,leucine, isoleucine, serine, threonine, or methionine and morepreferably valine or glycine and position 30 is glutamic acid, asparticacid, serine, or histidine and more preferably glutamic acid.

Other preferred GLP-1 analogs have the sequence of GLP-1(7-37)OH exceptthat the amino acid at position 8 is preferably glycine, valine,leucine, isoleucine, serine, threonine, or methionine and morepreferably valine or glycine and position 37 is histidine, lysine,arginine, threonine, serine, glutamic acid, aspartic acid, tryptophan,tyrosine, phenylalanine and more preferably histidine.

In a preferred embodiment, the GLP-1 analog is GLP-1(7-37)OH wherein theamino acid at position 12 is selected from the group consisting oftryptophan or tyrosine. It is more preferred that in addition to thesubstitution at position 12, the amino acid at position 8 is substitutedwith glycine, valine, leucine, isoleucine, serine, threonine, ormethionine and more preferably valine or glycine. It is even morepreferred that in addition to the substitutions at position 12 and 8,the amino acid at position 22 is substituted with glutamic acid.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 16 is selected from the groupconsisting of tryptophan, isoleucine, leucine, phenylalanine, ortyrosine. It is more preferred that in addition to the substitution atposition 16, the amino acid at position 8 is substituted with glycine,valine, leucine, isoleucine, serine, threonine, or methionine and morepreferably valine or glycine. It is even more preferred that in additionto the substitutions at position 16 and 8, the amino acid at position 22is substituted with glutamic acid. It is also preferred that in additionto the substitutions at positions 16 and 8, the amino acid at position30 is substituted with glutamic acid. It is also preferred that inaddition to the substitutions at positions 16 and 8, the amino acid atposition 37 is substituted with histidine.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 18 is selected from the groupconsisting of tryptophan, tyrosine, phenylalanine, lysine, leucine, orisoleucine, preferably tryptophan, tyrosine, and isoleucine. It is morepreferred that in addition to the substitution at position 18, the aminoacid at position 8 is substituted with glycine, valine, leucine,isoleucine, serine, threonine, or methionine and more preferably valineor glycine. It is even more preferred that in addition to thesubstitutions at position 18 and 8, the amino acid at position 22 issubstituted with glutamic acid. It is also preferred that in addition tothe substitutions at positions 18 and 8, the amino acid at position 30is substituted with glutamic acid. It is also preferred that in additionto the substitutions at positions 18 and 8, the amino acid at position37 is substituted with histidine.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 19 is selected from the groupconsisting of tryptophan or phenylalanine, preferably tryptophan. It ismore preferred that in addition to the substitution at position 19, theamino acid at position 8 is substituted with glycine, valine, leucine,isoleucine, serine, threonine, or methionine and more preferably valineor glycine. It is even more preferred that in addition to thesubstitutions at position 19 and 8, the amino acid at position 22 issubstituted with glutamic acid. It is also preferred that in addition tothe substitutions at positions 19 and 8, the amino acid at position 30is substituted with glutamic acid. It is also preferred that in additionto the substitutions at positions 19 and 8, the amino acid at position37 is substituted with histidine.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 20 is phenylalanine, tyrosine, ortryptophan. It is more preferred that in addition to the substitution atposition 20, the amino acid at position 8 is substituted with glycine,valine, leucine, isoleucine, serine, threonine, or methionine and morepreferably valine or glycine. It is even more preferred that in additionto the substitutions at position 20 and 8, the amino acid at position 22is substituted with glutamic acid. It is also preferred that in additionto the substitutions at positions 20 and 8, the amino acid at position30 is substituted with glutamic acid. It is also preferred that inaddition to the substitutions at positions 20 and 8, the amino acid atposition 37 is substituted with histidine.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 25 is selected from the groupconsisting of valine, isoleucine, and leucine, preferably valine. It ismore preferred that in addition to the substitution at position 25, theamino acid at position 8 is substituted with glycine, valine, leucine,isoleucine, serine, threonine, or methionine and more preferably valineor glycine. It is even more preferred that in addition to thesubstitutions at position 25 and 8, the amino acid at position 22 issubstituted with glutamic acid. It is also preferred that in addition tothe substitutions at positions 25 and 8, the amino acid at position 30is substituted with glutamic acid. It is also preferred that in additionto the substitutions at positions 25 and 8, the amino acid at position37 is substituted with histidine.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 27 is selected from the groupconsisting of isoleucine or alanine. It is more preferred that inaddition to the substitution at position 27, the amino acid at position8 is substituted with glycine, valine, leucine, isoleucine, serine,threonine, or methionine and more preferably valine or glycine. It iseven more preferred that in addition to the substitutions at position 27and 8, the amino acid at position 22 is substituted with glutamic acid.It is also preferred that in addition to the substitutions at positions27 and 8, the amino acid at position 30 is substituted with glutamicacid. It is also preferred that in addition to the substitutions atpositions 27 and 8, the amino acid at position 37 is substituted withhistidine.

In another preferred embodiment, the GLP-1 analog is GLP-1(7-37)OHwherein the amino acid at position 33 is isoleucine. It is morepreferred that in addition to the substitution at position 33, the aminoacid at position 8 is substituted with glycine, valine, leucine,isoleucine, serine, threonine, or methionine and more preferably valineor glycine. It is even more preferred that in addition to thesubstitutions at position 33 and 8, the amino acid at position 22 issubstituted with glutamic acid. It is also preferred that in addition tothe substitutions at positions 33 and 8, the amino acid at position 30is substituted with glutamic acid. It is also preferred that in additionto the substitutions at positions 33 and 8, the amino acid at position37 is substituted with histidine.

It is preferable that the GLP-1 compounds of formula I, II, III, V, V,or VI have 6 or fewer changes compared to the corresponding amino acidsin native GLP-1(7-37)OH.

More preferred analogs have 5 or fewer changes compared to thecorresponding amino acids in native GLP-1(7-37)OH or have 4 or fewerchanges compared to the corresponding amino acids in nativeGLP-1(7-37)OH or have 3 changes compared to the corresponding aminoacids in native GLP-1(7-37)OH.

Some preferred GLP-1 compounds of formula I, II, III, IV, V, and VIhaving multiple substitutions include GLP-1(7-37)OH wherein position 8is valine or glycine, position 22 is glutamic acid, position 16 istyrosine, leucine or tryptophan, position 18 is tyrosine, tryptophan, orisoleucine, position 25 is valine and position 33 is isoleucine. Otherpreferred GLP-1 compounds include the following:Val⁸-Tyr¹⁶-GLP-1(7-37)OH, Val⁸-Tyr¹²-Glu²²-GLP-1(7-37)OH,Val⁸-Tyr¹⁶-Phe¹⁹-GLP-1(7-37)OH, Val⁸-Tyr¹⁶-Glu²²-GLP-1(7-37)OH,Val⁸-Trp¹⁶-Glu²²-GLP-1(7-37)OH, Val⁸-Leu¹⁶-Glu²²-GLP-1(7-37)OH,Val⁸-Ile¹⁶-Glu²²-GLP-1(7-37)OH, Val⁸-Phe¹⁶-Glu²²-GLP-1(7-37)OH,Val⁸-Trp¹⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Tyr¹⁸-Glu²²-GLP-1(7-37)OH,Val⁸-Phe¹⁸-Glu²²-GLP-1(7-37)OH, and Val⁸-Ile¹⁸-Glu²²-GLP-1(7-37)OH.

Substitutions at the positions disclosed herein result in a GLP-1compound with increased potency compared to the potency ofVal⁸-GLP-1(7-37)OH. The GLP-1 compounds of the present inventiongenerally are between 3 and 6-fold more potent than Val⁸-GLP-1(7-37)OH.For example, table 8 and 9 provide a list of GLP-1 compounds with an invitro potency compared to that obtained for GLP-1(7-37)OH andVal⁸-GLP-1(7-37)OH, respectively. Preferably, the analogs are greaterthan 3-fold more potent than GLP-1(7-37)OH or Val⁸-GLP-1(7-37)OH. The invitro potencies disclosed in table 8 and 9 are generally representativeof in vivo potency relative to GLP-1(7-37)OH or Val⁸-GLP-1(7-37)OH.

Furthermore, many of these more potent analogs have a reduced propensityto aggregate and thus, have increased stability. GLP-1 compounds canexist in at least two different forms. The first form is physiologicallyactive and dissolves readily in aqueous solution at physiological pH(7.4). A second inactive form is readily produced when aqueous GLP-1solutions are agitated, exposed to hydrophobic surfaces or have largeair/water interfaces. The tendency to convert to the insoluble formconsiderably complicates the production of commercial quantities ofactive GLP-1 compounds. Thus, GLP-1 compounds that have a reducedpropensity to aggregate in solution and are more potent thanGLP-1(7-37)OH or Val⁸-GLP-1(7-37)OH are preferred. For example, theGLP-1 compounds Val⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Glu³⁰-GLP-1(7-37)OH, andVal⁸-His³⁷-GLP-1(7-37)OH show a markedly decreased propensity toaggregate in solution compared to GLP-1(7-37)OH or Val⁸-GLP-1(7-37)OH(See tables 8 and 9). Thus, preferred GLP-1 compounds of the presentinvention have either a glutamic acid at position 22, a glutamic acid atposition 30, or a histidine at position 37 or combinations thereof inaddition to substitutions at other positions such as 12, 16, 18, 19, 20,25, 27, and 33.

Preferred embodiments of formula I, II, III, IV, V, and VI include GLP-1compounds that do not differ from GLP-1(7-34)OH or GLP-1(7-36)NH₂ bymore than 6 amino acids, by more than 5 amino acids, by more than 4amino acids, or by more than 3 amino acids. It is also preferable thatthe GLP-1 compounds of formula I, II, I, IV, V, and VI have valine orglycine at position 8 and glutamic acid at position 22. It is alsopreferable that the GLP-1 compounds of formula I and II have valine orglycine at position 8 and glutamic acid at position 30. It is alsopreferable that the GLP-1 compounds of formula I, II, III, IV, V, and VIhave valine or glycine at position 8 and histidine at position 37.

Examples of GLP-1 analogs that have been shown to have a markedlydecreased propensity to aggregate compared with GLP-1(7-37)OH aredescribed in examples 8 and Table 9. Examples of GLP-1 analogs that havebeen shown to have a markedly increased GLP-1 receptor activation aredescribed in Example 9 and Tables 8 and 9. Preferably the GLP-1 analogsof the present invention are described in Table 8 and Table 9.

GLP-1 compounds of the present invention also include GLP-1 derivatives.A “GLP-1 derivative” refers an amino acid sequence of naturallyoccurring truncated GLP-1 compounds, GLP-1 fragments, or a GLP-1 analog,but additionally having a chemical modification of one or more of itsamino acid side groups, α-carbon atoms, terminal ammo group, or terminalcarboxylic acid group. A chemical modification includes, but is notlimited to, adding chemical moieties, creating new bonds, and removingchemical moieties. Modifications at amino acid side groups include,without limitation, acylation of lysine ε-amino groups, N-alkylation ofarginine, histidine, or lysine, alkylation of glutamic or asparticcarboxylic acid groups, and deamidation of glutamine or asparagine.Modifications of the terminal amino group include, without limitation,the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include,without limitation, the amide, lower alkyl amide, dialkyl amide, andlower allyl ester modifications. Furthermore, one or more side groups,or terminal groups, may be protected by protective groups known to theordinarily-skilled protein chemist. The α-carbon of an amino acid may bemono- or dimethylated. Preferred GLP-1 derivatives are described in U.S.Pat. No. 6,268,343 B1. A more preferred GLP-1 derivative isArg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37).

Preferred GLP-1 compounds that may be derivitized include GLP-1 analogshaving modifications at one or more of the following positions: 8, 12,16, 18, 19, 20, 22, 25, 27, 30, 33, and 37 and show increased potencycompared with Val⁸-GLP-1(7-37)OH.

Other preferred GLP-1 compounds that may be derivitized are analogs ofExendin-3 or Exendin-4. These GLP-1 compounds are described inWO99/07404, WO99/25727, WO99/25728, WO99/43708, WO00/66629, andUS2001/0047084A1 and are herein incorporated by reference.

Representative examples of basal insulins that can be useful in thepresent invention are known in the art and are commercially available.An example of a basal insulin is insulin-NPH. Insulin-NPH is prepared bytechniques widely accepted in the art and described U.S. Pat. No.5,547,929 and Hagedorn, H. C., et al., (1936) J. Am. Med. Assn. 106,177-180 and are herein incorporated by reference.

Another example of a basal insulin is an NPH-like preparation of amonomeric insulin analog referred to as monomeric insulin analog-NPD,more commonly known in the art as “NPL”. NPL is Neutral Protamineformulation of LysB28-ProB29 wherein the formulation comprises aninsulin complex comprising LysB28-ProB29 complexed with protamine andzinc in an aqueous medium. NPL is described in U.S. Pat. Nos. 5,461,031,5,650,486, and 5,747,642.

The insulin analogs used in the present invention can be prepared by anyof a variety of recognized peptide synthesis techniques includingclassical solution methods, solid phase methods, semi-synthetic methods,and recombinant DNA methods. Chance, et al., U.S. Pat. No. 5,514,646,issued May 7, 1996, discloses the preparation of various insulin analogswith sufficient detail to enable one skilled in the art to prepare anyof the insulin analogs used in the present invention.

AspB28 protamine insulin crystals represent another basal insulin.AspB28 protamine insulin crystal formulations according to Balschmidtare described in U.S. Pat. No. 5,840,680. The formulations comprisecrystals comprising AspB28, protamine, and a member selected from thegroup consisting of phenol, m-cresol, or a combination thereof andoptionally further comprising zinc, in an aqueous medium.

Additionally, it is possible to have a basal insulin formulation thatcomprises different insulins that would be useful in the presentinvention. Brader described in WO99/32116 co-crystals comprising aderivatized insulin, an un-derivatized insulin, zinc, protamine, and aphenolic preservative that provide flexibility of control over theduration and shape of the glucodynamic response profile.

There are a number of other basal insulin formulations that can also beuseful in the present invention. These formulations include but are notlimited to insulin zinc suspensions (Ultralente (UL) and Semilenteinsulins) and protamine zinc insulin (PZI). Preparation of these basalinsulins are well known in the art and described by Brange, in Galenicsoflinsulin: The Physico-chemical and Pharmaceutical Aspects of Insulinand Insulin Preparation, Springer-Verlag Berlin Heidelbert (1987).

Other basal insulin formulations useful in the present invention aregenerally described as soluble acylated basal insulins. Theseformulations include but are not limited to insulins acylated by fattyacids in the epsilon (ε) amino group of LysB29.

Preparation of these basal insulins are well known in the art anddescribed by Meyers, et al., (1997) Diabetes 46, 637-642, by Markussen,et al., (1996) Diabetologia 39, 281-288, and Havelund, et al.,WO95/07931. Acylated insulins or insulin analogs are further describedin U.S. Pat. No. 6,011,007. These soluble basal insulins comprise alipophilic substituent at the ε-amino group of the lysine at position 29of the B chain. In one embodiment, acylated insulins analogs can haveany amino acid at position 21 of the A chain except Lys, Arg and Cys,can have any amino acid at position 3 of the B chain except Lys, Arg andCys, and can have the Phe at position 1 of the B chain present ordeleted. Position 30 of the B chain can be (a) a non-codable, lipophilicamino acid having from 10 to 24 carbon atoms, in which case an acylgroup of a carboxylic acid with up to 5 carbon atoms is bound to theepsilon-amino group of Lys at position 29 of the B chain, (b) any aminoacid except Lys, Arg and Cys, in which case the epsilon-amino group ofLys at position 29 of the B chain has a lipophilic substituent or (c)deleted, in which case the epsilon-amino group of Lys at position 29 ofthe B chain has a lipophilic substituent; and any Zn⁺⁺ complexesthereof, provided that when position 30 of the B chain is Thr or Ala,positions 21 of the A chain and position 3 of the B chain are both Asn,and position 1 of the B chain is Phe, then the acylated insulin analogis a Zn⁺⁺ complex. Examples of acylated insulin analogs are described inTable 1.

TABLE 1 NεB29-tridecanoyl des(B30) human insulin, NεB29-tetradecanoyldes(B30) human insulin, NεB29-decanoyl des(B30) human insulin,NεB29-dodecanoyl des(B30) human insulin, NεB29-tridecanoyl GlyA21des(B30) human insulin, NεB29-tetradecanoyl GlyA21 des(B30) humaninsulin, NεB29-decanoyl GlyA21 des(B30) human insulin, NεB29-dodecanoylGlyA21 des(B30) human insulin, NεB29-tridecanoyl GlyA21 GlnB3 des(B30)human insulin, NεB29-tetradecanoyl GlyA21 GlnB3 des(B30) human insulin,NεB29-decanoyl GlyA21 GlnB3 des(B30) human insulin, NεB29-dodecanoylGlyA21 GlnB3 des(B30) human insulin, NεB29-tridecanoyl AlaA21 des(B30)human insulin, NεB29-tetradecanoyl AlaA21 des(B30) human insulin,NεB29-decanoyl AlaA21 des(B30) human insulin, NεB29-dodecanoyl AlaA21des(B30) human insulin, NεB29-tridecanoyl AlaA21 GlnB3 des(B30) humaninsulin, NεB29-tetradecanoyl AlaA21 GlnB3 des(B30) human insulin,NεB29-decanoyl AlaA21 GlnB3 des(B30) human insulin, NεB29-dodecanoylAlaA21 GlnB3 des(B30) human insulin, NεB29-tridecanoyl GlnB3 des(B30)human insulin, NεB29-tetradecanoyl GlnB3 des(B30) human insulin,NεB29-decanoyl GlnB3 des(B30) human insulin, NεB29-dodecanoyl GlnB3des(B30) human insulin, NεB29-tridecanoyl GlyA21 human insulin,NεB29-tetradecanoyl GlyA21 human insulin, NεB29-decanoyl GlyA21 humaninsulin, NεB29-dodecanoyl GlyA21 human insulin, NεB29-tridecanoyl GlyA21GlnB3 human insulin, NεB29-tetradecanoyl GlyA21 GlnB3 human insulin,NεB29-decanoyl GlyA21 GlnB3 human insulin, NεB29-dodecanoyl GlyA21 GlnB3human insulin, NεB29-tridecanoyl AlaA21 human insulin,NεB29-tetradecanoyl AlaA21 human insulin, NεB29-decanoyl AlaA21 humaninsulin, NεB29-dodecanoyl AlaA21 human insulin, NεB29-tridecanoyl AlaA21GlnB3 human insulin, NεB29-tetradecanoyl AlaA21 GlnB3 human insulin,NεB29-decanoyl AlaA21 GlnB3 human insulin, NεB29-dodecanoyl AlaA21 GlnB3human insulin, NεB29-tridecanoyl GlnB3 human insulin,NεB29-tetradecanoyl GlnB3 human insulin, NεB29-decanoyl GlnB3 humaninsulin, NεB29-dodecanoyl GlnB3 human insulin, NεB29-tridecanoyl GluB30human insulin, NεB29-tetradecanoyl GluB30 human insulin, NεB29-decanoylGluB30 human insulin, NεB29-dodecanoyl GluB30 human insulin,NεB29-tridecanoyl GlyA21 GluB30 human insulin, NεB29-tetradecanoylGlyA21 GluB30 human insulin, NεB29-decanoyl GlyA21 GluB30 human insulin,NεB29-dodecanoyl GlyA21 GluB30 human insulin, NεB29-tridecanoyl GlyA21GlnB3 GluB30 human insulin, NεB29-tetradecanoyl GlyA21 GlnB3 GluB30human insulin, NεB29-decanoyl GlyA21 GlnB3 GluB30 human insulin,NεB29-dodecanoyl GlyA21 GlnB3 GluB30 human insulin, NεB29-tridecanoylAlaA21 GluB30 human insulin, NεB29-tetradecanoyl AlaA21 GluB30 humaninsulin, NεB29-decanoyl AlaA21 GluB30 human insulin, NεB29-dodecanoylAlaA21 GluB30 human insulin, NεB29-tridecanoyl AlaA21 GlnB3 GluB30 humaninsulin, NεB29-tetradecanoyl AlaA21 GlnB3 GluB30 human insulin,NεB29-decanoyl AlaA21 GlnB3 GluB30 human insulin, NεB29-dodecanoylAlaA21 GlnB3 GluB30 human insulin, NεB29-tridecanoyl GlnB3 GluB30 humaninsulin, NεB29-tetradecanoyl GlnB3 GluB30 human insulin, NεB29-decanoylGlnB3 GluB30 human insulin and NεB29-dodecanoyl GlnB3 GluB30 humaninsulin.

Other basal insulin formulations useful in the present invention aregenerally described as soluble pI shifted basal insulin analogs. pIshifted analogs are described in U.S. Pat. No. 5,656,722. These analogshave pls between about 5 and about 8.5 as a result of basicmodifications to the amino acid sequence. They are stable at the weaklyacid pH values of appropriate formulations even for extended periods. pIshifted analogs of human insulin have a basic modification on theC-terminal end of the B chain, a substitution of the asparagine atposition 21 of the A chain, and where appropriate, a substitution of thehistidine at position 10 of the B chain. Preferably, the pI shiftedinsulin analog has Gly at position 21 of the A chain and Arg-Arg-OH atposition 31 of the B chain. Examples of pI shifted insulin analogs aredescribed in Table 2.

TABLE 2 AspA21-Human insulin-ArgB31-OH GluA21-Human insulin-ArgB31-OHGlyA21-Human insulin-ArgB31-OH SerA21-Human insulin-ArgB31-OHThrA21-Human insulin-ArgB31-OH AlaA21-Human insulin-ArgB31-OHAspA21-Human insulin-ArgB31-ArgB32-OH GluA21-Humaninsulin-ArgB31-ArgB32-OH GlyA21-Human insulin-ArgB31-ArgB32-OHSerA21-Human insulin-ArgB31-ArgB32-OH ThrA21-Humaninsulin-ArgB31-ArgB32-OH AlaA21-Human insulin-ArgB31-ArgB32-OHAspA21-AsnB10-Human insulin-ArgB31-OH GluA21-AsnB10-Humaninsulin-ArgB31-OH GlyA21-AsnB10-Human insulin-ArgB31-OHSerA21-AsnB10-Human insulin-ArgB31-OH ThrA21-AsnB10-Humaninsulin-ArgB31-OH AlaA21-AsnB10-Human insulin-ArgB31-OHAspA21-AsnB10-Human insulin-ArgB31-ArgB32-OH GluA21-AsnB10-Humaninsulin-ArgB31-ArgB32-OH GlyA21-AspB10-Human insulin-ArgB31-ArgB32-OHSerA21-AsnB10-Human insulin-ArgB31-ArgB32-OH ThrA21-AsnB10-Humaninsulin-ArgB31-ArgB32-OH AlaA21-AsnB10-Human insulin-ArgB31-ArgB32-OH

Other soluble pI shifted basal insulin analogs useful in the presentinvention are described in Markussen, et al., Protein Eng. (1988) 2:157.A preferred analog described by Markussen, et al. isGlyA21ArgB27ThrB30-NH₂. This analog is generally known as NovoSol Basal.

In another embodiment, the pI shifted insulin analog is human insulinwith Arg at position 0 of the A chain, and Arg attached to the epsilonamino group of the Lys at position 29 of the B chain(A0^(Arg)-B29^(Lys-Nε-Arg)). In another embodiment, the pI shiftedinsulin analog is human insulin with Arg at position 0 of the A chain,any amino acid substituted for Asn at position 21 of the A chain, andArg attached to the epsilon amino group of the Lys at position 29 of theB chain (A0^(Arg)-A21^(Xaa)-B29^(Lys-Nε-Arg)). In another embodiment,the pI shifted insulin analog is human insulin with Arg at position 0 ofthe A chain, Arg at position 0 of the B chain, and Arg attached to theepsilon amino group of the Lys at position 29 of the B chain(A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg)). In another embodiment, the pIshifted insulin analog is human insulin with Arg at position 0 of the Achain, any amino acid substituted for Asn at position 21 of the A chain,Arg at position 0 of the B chain, and Arg attached to the epsilon aminogroup of the Lys at position 29 of the B chain(A0^(Arg)-A21^(Xaa)-B0^(Arg)-B29^(Lys-Nε-Arg)). In another embodiment,the pI shifted insulin analog human insulin with Lys with Arg attachedto its epsilon amino group at position at position 0 of the A chain, andArg attached to the epsilon amino group of the Lys at position 29 of theB chain (A0^(Lys-Nε-Arg)-B29^(Lys-Nε-Arg). In another embodiment, the pIshifted insulin analog human insulin with Lys with Arg attached to itsepsilon amino group at position at position 0 of the A chain, any aminoacid substituted for Asn at position 21 of the A chain, and Arg attachedto the epsilon amino group of the Lys at position 29 of the B chain(A0^(Lys-Nε-Arg)-A21^(Xaa)-B29^(Lys-Nε-Arg)).

Other soluble pI shifted basal insulin analogs are described in UnitedStates Provisional application by Beals et al. entitled Insulin MoleculeHaving Protracted Time Action, filed on Aug. 2, 2002, at the UnitedStates Patent and Trademark Office. Examples of these pI shifted basalinsulin analogs are described in Table 3.

TABLE 3 A0^(Arg)B0^(Arg)-insulin; A0^(Arg)B0^(Arg)A21^(Xaa)-insulin;A0^(Arg)B0^(Arg)A21^(Gly)-insulin; A0^(Arg)B0^(Arg)A21^(Ser)-insulin;A0^(Lys)B0^(Lys)-insulin; A0^(Lys)B0^(Lys)A21^(Xaa)-insulin;A0^(Lys)B0^(Lys)A21^(Gly)-insulin; A0^(Lys)B0^(Lys)A21^(Ser)-insulin;A0^(Arg)B0^(Lys)-insulin; A0^(Arg)B0^(Lys)A21^(Xaa)-insulin;A0^(Arg)B0^(Lys)A21^(Gly)-insulin; A0^(Arg)B0^(Lys)A21^(Ser)-insulin;A0^(Lys)B0^(Arg)-insulin; A0^(Lys)B0^(Arg)A21^(Xaa)-insulin;A0^(Lys)B0^(Arg)A21^(Gly)-insulin; A0^(Lys)B0^(Arg)A21^(Ser)-insulin;A0^(Arg)B0^(Arg)B28^(Lys-Nε-Arg)B29^(Pro)-insulin;A0^(Arg)A21^(Xaa)B28^(Lys-Nε-Arg)B29^(Pro)-insulin;A0^(Arg)A21^(Gly)B28^(Lys-Nε-Arg)B29^(Pro)-insulin;A0^(Arg)A21^(Gly)B28^(Lys-Nε-Arg)B29^(Pro)-insulin;A0^(Arg)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)A21^(Gly)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)B29^(Lys-Nε-Lys)-insulin;A0^(Arg)A21^(Xaa)B29^(Lys-Nε-Lys)-insulin;A0^(Arg)A21^(Gly)B29^(Lys-Nε-Lys)-insulin;A0^(Arg)A21^(Ser)B29^(Lys-Nε-Lys)-insulin;A-1^(Arg)A0^(Lys)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Lys)A21^(Gly)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Lys)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Lys)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Lys)A21^(Gly)B29^(Lys-Nε-Arg)insulin;A-1^(Arg)A0^(Lys)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A-1^(Lys)A0^(Lys)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A-1^(Lys)A0^(Lys)A21^(Gly)B29^(Lys-Nε-Arg)-insulin;A-1^(Lys)A0^(Lys)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A-1^(Lys)A0^(Lys)A21^(Xaa)B29^(Lys-Nε-Lys)-insulin;A-1^(Lys)A0^(Lys)A21^(Gly)B29^(Lys-Nε-Lys)insulin;A-1^(Lys)A0^(Lys)A21^(Ser)B29^(Lys-Nε-Lys)-insulin;A-1^(Arg)A0^(Arg)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Arg)A21^(Gly)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Arg)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A-1^(Arg)A0^(Arg)A21^(Xaa)B29^(Lys-Nε-Lys)-insulin;A-1^(Arg)A0^(Arg)A21^(Gly)B29^(Lys-Nε-Lys)insulin;A-1^(Arg)A0^(Arg)A21^(Ser)B29^(Lys-Nε-Lys)-insulin;A0^(Lys-Nε-Arg)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A0^(Lys-Nε-Arg)A21^(Gly)B29^(Lys-Nε-Arg)-insulin;A0^(Lys-Nε-Arg)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A0^(Lys-Nε-Arg)A21^(Xaa)B29^(Lys-Nε-Lys)-insulin;A0^(Lys-Nε-Arg)A21^(Gly)B29^(Lys-Nε-Lys)insulin;A0^(Lys-Nε-Arg)A21^(Ser)B29^(Lys-Nε-Lys)-insulin;A0^(Lys-Nε-Lys)A21^(Xaa)B29^(Lys-Nε-Arg)-insulin;A0^(Lys-Nε-Lys)A21^(Gly)B29^(Lys-Nε-Arg)-insulin;A0^(Lys-Nε-Lys)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A0^(Lys-Nε-Lys)A21^(Xaa)B29^(Lys-Nε-Lys)-insulin;A0^(Lys-Nε-Lys)A21^(Gly)B29^(Lys-Nε-Lys)insulin;A0^(Lys-Nε-Lys)A21^(Ser)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)B0^(Arg)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)A21^(Xaa)B0^(Arg)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)A21^(Gly)B0^(Arg)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)A21^(Ser)B0^(Arg)B29^(Lys-Nε-Arg)-insulin;A0^(Arg)B0^(Arg)B29^(Lys-Nε-Lys)-insulin;A0^(Arg)A21^(Xaa)B0^(Arg)B29^(Lys-Nε-Lys)-insulin;A0^(Arg)A21^(Gly)B0^(Arg)B29^(Lys-Nε-Lys)-insulin;A0^(Arg)A21^(Ser)B0^(Arg)B29^(Lys-Nε-Lys)-insulin;A0^(Lys)B0^(Lys)B29^(Lys-Nε-Arg)-insulin;A0^(Lys)A21^(Xaa)B0^(Lys)B29^(Lys-Nε-Arg)-insulin;A0^(Lys)A21^(Gly)B0^(Lys)B29^(Lys-Nε-Arg)-insulin;A0^(Lys)A21^(Ser)B0^(Lys)B29^(Lys-Nε-Arg)-insulin;A0^(Lys)B0^(Lys)B29^(Lys-Nε-Lys)-insulin;A0^(Lys)A21^(Xaa)B0^(Lys)B29^(Lys-Nε-Lys)-insulin;A0^(Lys)A21^(Gly)B0^(Lys)B29^(Lys-Nε-Lys)-insulin;A0^(Lys)A2^(Ser)B0^(Lys)B29^(Lys-Nε-Lys)-insulin;A0^(gHR)B0^(gHR)B29^(Lys-Nε-Arg)-insulin;A0^(gHR)A21^(Xaa)B0^(gHR)B29^(Lys-Nε-Arg)-insulin;A0^(gHR)A21^(Gly)B0^(gHR)B29^(Lys-Nε-Arg)-insulin; andA0^(gHR)A21^(Ser)B0^(gHR)B29^(Lys-Nε-Arg)-insulin.

Both GLP-1 compounds and the insulins can have an acid at the C-terminalor an amide at the C-terminal. Native GLP-1 is an amide, therefore it ispreferred that the GLP-1 compounds of the present invention also beamides. Native human insulin however, is an acid, therefore, it ispreferred that the insulins of the present invention also be acids.

The present invention further provides a process of preparing pre-mixedformulations by mixing a GLP-1 compound with a basal insulin. Basalinsulin may be added as a solid to a solution containing a GLP-1compound or solid GLP-1 compound may be dissolved in a suspension orsolution containing basal insulin. Alternatively, both the basal insulinand the GLP-1 compound may be added in any order to a buffered solution.In one embodiment, stock solutions of the GLP-1 compound and the basalinsulin be prepared separately and then added together at the desiredratio. Preferably, a 100 Unit/ml basal insulin suspension or solution isdiluted with a GLP-1 stock solution such that the final pre-mixedformulation has between 30 units/ml and 70 units/ml of basal insulin. Inanother embodiment, solid GLP-1 compound is dissolved in a suspension orsolution containing the basal insulin. Preferably, the solid GLP-1 isdissolved in a 100 Unit/ml basal insulin suspension or solution suchthat the 100 Unit/ml concentration of basal insulin is maintained in themixture formulation. Preferably, the pre-mixed formulations are bufferedand contain a preservative and an isotonicity agent. The GLP-1 compoundand basal insulin must be mixed in such a way as to preserve theirbiological activity and time-action.

For example, a pre-mixed formulation containing NPH or NPL can beprepared by dissolving a GLP-1 compound in NPH or NPL diluent. Theexcipients and pH of the diluent may be adjusted to maintain the GLP-1compound in solution. However, the pH or concentration of buffer orother excipients cannot be such that when the GLP-1 compound solution isadded to NPH or NPL, some or all of the insulin dissolves. Thus, it ispreferable that the final pH of a pre-mixed formulation comprised of NPHor NPL be between about 7 and about 8, preferably 7.2 and 7.8. Differentvolumes of the GLP-1 compound solution can then be added to NPH or NPLsuch that the desired ratio of GLP-1 compound to insulin is obtained.

The following examples are provided merely to further illustrate thepreparation of the pre-mixed formulations of the invention. The scope ofthe invention is not construed as merely consisting of the followingexamples.

EXAMPLE 1

Insulinotropic Activity Determination:

A collagenase digest of pancreatic tissue is separated on a Ficollgradient (27%, 23%, 20.5%, and 11% in Hank's balanced salt solution, pH7.4). The islets are collected from the 20.5%/11% interface, washed andhandpicked free of exocrine and other tissue under a stereomicroscope.The islets are incubated overnight in RPMI 1640 medium supplemented with10% fetal bovine plasma and containing 11 mM glucose at 37° C. and 95%air/5% CO₂. The GLP-1 compound to be studied is prepared at a range ofconcentrations, preferably 3 nanomolar to 30 nanomolar in RPMI mediumcontaining 10% fetal bovine plasma and 16.7 mM glucose. About 8 to 10isolated islets are then transferred by pipette to a total volume of 250μl of the GLP-1 compound containing medium in 96 well microtiter dishes.The islets are incubated in the presence of the GLP-1 compound at 37°C., 95% air, 5% CO₂ for 90 minutes. Then aliquots of islet-free mediumare collected and 100 μl thereof are assayed for the amount of insulinpresent by radioimmunoassay using an Equate Insulin RIA Kit (Binax,Inc., Portland, Me.).

EXAMPLE 2

Preparation of Val⁸-GLP-1 Solutions: Several Val⁸-GLP-1 solutions wereprepared to determine the conditions that maintained solubility of theVal⁸-GLP-1 in the presence of preservative systems used for typicalbasal insulin formulations. The samples were analyzed by reversed-phaseHPLC chromatography to determine the total protein concentrations andthe soluble protein concentrations. Table 4 summarizes the data from thevarious Val⁸-GLP-1 solutions.

TABLE 4 Theoretical Total Soluble Physical Val⁸-GLP-1 Protein ProteinPurity Buffer Appearance pH Conc. (mg/mL) Conc. (mg/mL) Conc. (mg/mL)(%) 20 mM TRIS clear 7.4 0.5 0.5 0.49 92.0 No Buffer clear 7.7 0.5 0.42— 92.1 14 mM clear 7.8 1.0 0.99 — 93.0 phosphate No Buffer clear 7.8 0.50.43 — 91.9 No Buffer clear 7.8 1.25 0.92 — N/A No Buffer clear 7.8 1.601.3 — 94.06 ave ave No Buffer clear 7.8 1.60 1.5 — N/A 14 mM clear 8.00.5 0.51 — 92.9 phosphate 14 mM clear 8.1 1.0 0.94 — 91.9 phosphate NoBuffer Cloudy 7.8 2.50 2.19 — N/A No Buffer Cloudy 7.8 5.0 4.01 — N/A 14mM Cloudy 7.4 1.0 0.96 — 96.8 phosphate 7 mM Cloudy 7.4 1.0 0.87 0.9193.4 phosphate 20 mM TRIS Cloudy 7.4 1.0 0.81 0.91 93.4 20 mM TRISCloudy 7.4 1.0 1.37 0.81 93.4

EXAMPLE 3

Insulin Complex:

Human insulin-NPH is prepared according to accepted practice as taughtby Krayenbiuhl and Rosenberg Crystalline Protamine Insulin 60-73,(1951). Alternatively, human insulin-NPH is commercially available in asuspension formulation under the name of Humulin-N™, manufactured by EliLilly and Company, Indianapolis, Ind. The unit dose and concentration ofNPH used as the stock supply in these experiments was U100 at 3.5 mg/mL.The pH of comnnercial vials of NPH was adjusted to 7.2-8.5 with HCl orNaOH. The samples were analyzed by reversed-phase HPLC chromatography todetermine the soluble protein concentrations. Table 5 summarizes thedata from the various NPH suspensions

TABLE 5 Sample Soluble Protein No. PH Conc. (mg/mL) 1 7.2 0.33 2 7.20.004 3 7.8 0.01 4 8.0 0.06 5 8.0 0.14 6 8.5 0.39 7 8.5 0.87

EXAMPLE 4

Val⁸GLP-1/NPH Mixtures:

A solution of Val⁸-GLP-1 was prepared by dissolving 160 mg Val⁸-GLP-1 in50 mL water while keeping the pH in the range of 10 to 10.5. TheVal⁸-GLP-1 is added in aliquots and the pH is monitored and adjustedwith base. After dissolution of Val⁸-GLP-1, the pH was adjusted to 8with HCl. The solution was then diluted to 100 mL 2×NPH diluent lackingphate buffer (3.2 mg/mL m-cresol, 1.30 mg/mL phenol and 32 mg/mLglycerin, pH 7.4) for a final Val⁸-GLP-1 concentration of 1.6 mg/mL. ThepH was adjusted to 7.8 with HCl or NaOH. The Val⁸-GLP-1 solution wasmixed with commercial insulin-NPH suspension (U100, 3.5 mg/mL) at ratiosof 30:70, 50:50, and 70:30 (volume:volume) Val⁸-GLP-1:NPH. TheVal⁸-GLP-1:NPH mixture contained final concentrations of 1.6 mg/mLm-cresol, 0.65 mg/mL phenol, 10 mM, 7 mM, and 4 mLM phosphate buffer for30:70, 50:50, and 70:30 ratios, respectively, and 16 mg/mL glycerin. ThepH was adjusted to a final pH of 7.8 with HCl or NaOH. The samples wereanalyzed by reversed-phase HPLC chromatography to determine the totalprotein concentrations and the soluble protein concentrations. Table 6summarizes the data from the various Val⁸-GLP-1/NPH mixtures.

TABLE 6 Total Soluble Theoretical Protein Protein Concentration Conc.Conc. Val⁸-GLP-1/ (mg/mL) (mg/mL) (mg/mL) NPH Val⁸⁻ Val⁸- Val⁸- RatioGLP-1 Insulin GLP-1 Insulin GLP-1 Insulin 30:70 0.48 2.45 0.45 2.44 0.020.11 30:70 0.48 2.45 0.29 2.30 0.01 0.07 30:70 0.48 2.45 0.30 2.26 0.010.14 50:50 0.8 1.75 0.53 1.71 0.06 0.41 50:50 0.8 1.75 0.51 1.67 0.030.48 50:50 0.8 1.75 0.50 1.68 0.03 0.41 70:30 1.12 1.05 0.92 1.09 0.150.68 70:30 1.12 1.05 0.92 1.04 0.09 0.86 70:30 1.12 1.05 0.71 1.06 0.130.73

The low soluble protein concentrations of Val⁸-GLP-1 suggest that theVal⁸-GLP-1 interacts with the NPH. However, despite adherence of theGLP-1 to the NPH crystals, the insulinotropic activity of Val⁸-GLP-1 isnot affected. See FIG. 4.

EXAMPLE 5

Exendin-4/NPH Mixtures:

A solution of Exendin-4 was prepared by dissolving 20 mg Exendin-4 in 5mL water. The pH was adjusted to 7.4 or 7.6 with HCl or NaOH. Thesolution was then diluted to 10 mL 2×NPH diluent lacking phosphatebuffer (3.2 mg/mL m-cresol, 1.30 mg/mL phenol and 32 mg/mL glycerin, pH7.4) for a final Exendin-4 concentration of 2.0 mg/mL. The pH wasadjusted to 7.4 with HCl or NaOH. The Exendin-4 solution was mixed withcommercial insulin-NPH suspension (U100, 3.5 mg/mL) at ratios of 30:70,50:50, and 70:30 (volume:volume) Exendin-4:NPH. The Exendin-4:NPHmixture contained final concentration of 1.6 mg/mL m-cresol, 0.65 mg/mLphenol, 10 mM, 7 mM, and 4 mM phosphate buffer for 30:70, 50:50, and70:30 ratios, respectively, and 16 mg/mL glycerin.

The pH was adjusted to a final pH of 7.6 with HCl or NaOH. The sampleswere analyzed by reversed-phase HPLC chromatography to determine thetotal protein concentrations and the soluble protein concentrations.Table 7 summarizes the data from the various Exendin-4/NPH mixtures.

TABLE 7 Total Soluble Theoretical Protein Protein Exendin-4/Concentration Conc. Conc. NPH Measured (mg/mL) (mg/mL) (mg/mL) Ratio pHExendin-4 Insulin Exendin-4 Insulin Exendin-4 Insulin 30:70 7.53 0.612.45 0.76 2.38 0.68 0.02 50:50 7.59 1.02 1.75 1.28 1.74 1.14 0.03 70:307.56 1.42 1.05 1.77 1.04 1.54 0.04

EXAMPLE 6

Exendin-4/Lantus® Mixtures:

A solution of Exendin-4 is prepared by dissolving 10 mg Exendin-4 in 5mL water. The pH is adjusted to 4 with HCl. The solution is then dilutedwith 5 mL 2× diluent (5.4 mg/mL m-cresol, 2.5 mg/mL NaAcetate and 32mg/mL mannitol) for a final Exendin-4 concentration of 1.0 mg/mL. The pHis adjusted to 4 with HCl. The Exendin-4 solution is mixed withcommercial Lantus® insulin (U100, 3.6378 mg/mL) at ratios of 30:70,50:50, and 70:30 (volume:volume) Exendin-4:Lantus®. The 30:70 mixture ofExendin-4:Lantus® contains final concentrations of 0.3 mg/mL Exendin-4,701U Lantus®, 2.7 mg/mL m-cresol, 0.75 mg/mL NaAcetate, 9.6 mg/mLmannitol, 21 μg/ml zinc, and 11.9 mg/ml glycerol. The pH is adjusted toa final pH of 4 with HCl or NaOH. The 50:50 mixture of Exendin-4:Lantus®contains final concentrations of 0.5 mg/mL Exendin-4, 50IU Lantus®, 2.7mg/mL m-cresol, 1.25 mg/mL NaAcetate, 16 mg/mL mannitol, 15 μg/ml zinc,and 8.5 mg/ml glycerol. The pH is adjusted to a final pH of 4 with HClor NaOH. The 70:30 mixture of Exendin-4:Lantus® contains finalconcentrations of 0.7 mg/mL Exendin-4, 30IU Lantus®, 2.7 mg/mL m-cresol,1.75 mg/mL NaAcetate, 22.4 mg/mL mannitol, 9 μg/ml zinc, and 5.1 mg/mlglycerol. The pH is adjusted to a final pH of 4 with HCl or NaOH.

EXAMPLE 7

Val⁸-Glu²²-GLP-1(7-37)OH/Lantus® Mixtures:

A solution of Val⁸-Glu²²-GLP-1(7-37)OH is prepared by dissolving 5 mgVal⁸-Glu²²-GLP-1(7-37)OH in 5 mL water. The pH is adjusted to 4 withHCl. The solution is then diluted with 5 mL 2× diluent (5.4 mg/mLm-cresol, 2.5 mg/mL NaAcetate and 32 mg/mL mannitol) for a finalVal⁸-Glu²²-GLP-1(7-37)OH concentration of 0.5 mg/mL. The pH is adjustedto 4 with HCl. The Val⁸-Glu²²-GLP-1(7-37)OH solution is mixed withcommercial Lantus® insulin (U00, 3.6378 mg/mL) at ratios of 30:70,50:50, and 70:30 (volume:volume) Val⁸-Glu²²-GLP-1(7-37)OH:Lantus®. The30:70 mixture of Val⁸-Glu²²-GLP-1(7-37)OH:Lantus® contains finalconcentrations of 0.15 mg/mL Val⁸-Glu²²-GLP-1(7-37)OH, 70IU Lantus®, 2.7mg/mL m-cresol, 0.75 mg/mL NaAcetate, 9.6 mg/mL mannitol, 21 μg/ml zinc,and 11.9 mg/ml glycerol. The pH is adjusted to a final pH of 4 with HClor NaOH. The 50:50 mixture of Val⁸-Glu²²-GLP-1(7-37)OH:Lantus® containsfinal concentrations of 0.25 mg/mL Val⁸-Glu²²-GLP-1(7-37)OH, 50IULantus®, 2.7 mg/mL m-cresol, 1.25 mg/mL NaAcetate, 16 mg/mL mannitol, 15μg/ml zinc, and 8.5 mg/ml glycerol. The pH is adjusted to a final pH of4 with HCl or NaOH. The 70:30 mixture ofVal⁸-Glu²²-GLP-1(7-37)OH:Lantus® contains final concentrations of 0.35mg/mL Val⁸-Glu²²-GLP-1(7-37)OH, 30IU Lantus®, 2.7 mg/mL m-cresol, 1.75mg/mL NaAcetate, 22.4 mg/mL mannitol, 9 μg/ml zinc, and 5.1 mg/mlglycerol. The pH is adjusted to a final pH of 4 with HCl or NaOH.

EXAMPLE 8

GLP Aggregation Assay:

GLP peptides can be analyzed with respect to their potential toaggregate in solution.

In general, peptides in solution are stirred at elevated temperature ina suitable buffer while recording turbidity at 350 nm as a function oftime. Time to the onset of aggregation is measured to quantify thepotential of a given GLP molecule to aggregate under these stressedconditions.

A GLP-1 compound is first dissolved under alkaline conditions (pH 10.5)for 30 minutes to dissolve any pre-aggregated material. The solution isthen adjusted to pH 7.4 and filtered. Specifically, 4 mg of alyophilized GLP-1 compound is dissolved in 3 ml of 10 mM phosphate/10 mMcitrate. The pH is adjusted to 10.0-10.5 and held for 30 minutes. Thesolution is adjusted with HCl to pH 7.4 and filtered through a suitablefilter, for example a Millex GV syringe filter (Millipore Corporation,Bedford, Mass.). This solution is then diluted to a final samplecontaining 0.3 mg/mL protein in 10 mM citrate, 10 mM phosphate, 150 mMNaCl, and adjusted to pH 7.4 to 7.5. The sample is incubated at 37° C.in a quartz cuvette. Every five minutes the turbidity of the solution ismeasured at 350 nm on an AVIV Model 14DS UV-VIS spectrophotometer(Lakewood, N.J.). For 30 seconds prior to and during the measurement thesolution is stirred using a magnetic stir bar from Stama Cells, Inc.(Atascadero, Calif.). An increase in OD at 350 nm indicates aggregationof the GLP-peptide. The time to aggregation is approximated by theintersection of linear fits to the pre-growth and growth phase accordingto method of Drake(Arvinte T, Cudd A, and Drake A F.(1993) J. Bio. Chem.268, 6415-6422).

The cuvette is cleaned between experiments with a caustic soap solution(e.g., Contrad-70).

The results for a number of GLP-1 compounds are reported in Table 8 asthe time in hours required for the compound to aggregate. As can beseen, these compounds show greatly increased aggregation times overother GLP-1 compounds.

EXAMPLE 9

GLP-1 Receptor Activation:

The ability of the GLP-1 compounds of the present invention to activatethe GLP-1 receptor was assessed using in vitro assays such as thosedescribed in EP 619,322 to Gelfand, et al., and U.S. Pat. No. 5,120,712,respectively. The entire teachings of these references are incorporatedherein by reference.

In vitro potency is the measure of the ability of a peptide to activatethe GLP-1 receptor in a cell-based assay. In vitro potency is expressedas the “EC₅₀” which is the effective concentration of compound thatresults in 50% activity in a single dose-response experiment. For thepurposes of the present invention, in vitro potency is determined usinga fluorescence assay that employs HEK-293 Aurora CRE-BLAM cells thatstably express the human GLP-1 receptor. These HEK-293 cells have stablyintegrated a DNA vector having a cAmp response element (CRE) drivingexpression of the β-lactamase (BLAM) gene. The interaction of a GLP-1agonist with the receptor initiates a signal that results in activationof the cAmp response element and subsequent expression of β-lactamase.The β-lactamase CCF2/AM substrate that emits fluorescence when it iscleaved by β-lactamase (Aurora Biosciences Corp.) can then be added tocells that have been exposed to a specific amount of GLP-1 agonist toprovide a measure of GLP-1 agonist potency. The assay is furtherdescribed in Zlokarnik, et al., (1998) Science 279:84-88 (See alsoExample 1). The EC₅₀ values were determined using the BLAM assaydescribed above by generating a dose response curve using dilutionsranging from 0.00003 nanomolar to 30 nanomolar. Relative in vitropotency values are established by running GLP-1(7-37)OH orVal⁸-GLP-1(7-37)OH as a control and assigning the control a referencevalue of 1.

The activity of these polypeptides relative to the activity ofGLP-1(7-37)OH or Val⁸-GLP-1(7-37)OH is reported in Tables 8 and 9.

TABLE 8 GLP-1 Receptor Activation GLP-1 Polypeptide (relative toGLP-1(7-37)OH) GLP-1(7-37)OH 1.0 Val⁸-GLP-1(7-37)OH 0.47Gly⁸-His¹¹-GLP-1(7-37)OH 0.282 Val⁸- Ala¹¹-GLP-1(7-37)OH 0.021Val⁸-Lys¹¹-GLP-1(7-37)OH 0.001 Val⁸-Tyr¹²-GLP-1(7-37)OH 0.81Val⁸-Glu¹⁶-GLP-1(7-37)OH 0.047 Val⁸-Ala¹⁶-GLP-1(7-37)OH 0.112Val⁸-Tyr¹⁶-GLP-1(7-37)OH 1.175 Val⁸-Lys²⁰-GLP-1(7-37)OH 0.33Gln²²-GLP-1(7-37)OH 0.42 Val⁸-Ala²²-GLP-1(7-37)OH 0.56Val⁸-Ser²²-GLP-1(7-37)OH 0.50 Val⁸-Asp²²-GLP-1(7-37)OH 0.40Val⁸-Glu²²-GLP-1(7-37)OH 1.29 Val⁸-Lys²²-GLP-1(7-37)OH 0.58Val⁸-Pro²²-GLP-1(7-37)OH 0.01 Val⁸-His²²-GLP-1(7-37)OH 0.14Val⁸-Lys²²-GLP-1(7-36)NH₂ 0.53 Val⁸-Glu²²-GLP-1(7-36)NH₂ 1.0Gly⁸-Glu²²-GLP-1(7-37)OH 1.07 Val⁸-Lys²³-GLP-1(7-37)OH 0.18Val⁸-His²⁴-GLP-1(7-37)OH 0.007 Val⁸-Lys²⁴-GLP-1(7-37)OH 0.02Val⁸-His²⁶-GLP-1(7-37)OH 1.6 Val⁸-Glu²⁶-GLP-1(7-37)OH 1.5Val⁸-His²⁷-GLP-1(7-37)OH 0.37 Val⁸-Ala²⁷-GLP-1(7-37)OH 0.47Gly⁸-Glu³⁰-GLP-1(7-37)OH 0.29 Val⁸-Glu³⁰-GLP-1(7-37)OH 0.29Val⁸-Asp³⁰-GLP-1(7-37)OH 0.15 Val⁸-Ser³⁰-GLP-1(7-37)OH 0.19Val⁸-His³⁰-GLP-1(7-37)OH 0.19 Val⁸-Glu³³-GLP-1(7-37)OH 0.039Val⁸-Ala³³-GLP-1(7-37)OH 0.1 Val⁸-Gly³³-GLP-1(7-37)OH 0.01Val⁸-Glu³⁴-GLP-1(7-37)OH 0.17 Val⁸-Pro³⁵-GLP-1(7-37)OH 0.094Val⁸-His³⁵-GLP-1(7-37)OH 0.41 Val⁸-Glu³⁵-GLP-1(7-37)OH 0.15Val⁸-Glu³⁶-GLP-1(7-37)OH 0.11 Val⁸-His³⁶-GLP-1(7-37)OH 0.22Val⁸-His³⁷-GLP-1(7-37)OH 0.33 Val⁸-Leu¹⁶-Glu²⁶-GLP-1(7-37)OH 0.23Val⁸-Lys²²-Glu³⁰-GLP-1(7-37)OH 0.37 Val⁸-Lys²²-Glu²³-GLP-1(7-37)OH 0.35Val⁸-Glu²²-Ala²⁷-GLP-1(7-37)OH 1.02 Val⁸-Glu²²-Lys²³-GLP-1(7-37)OH 1.43Val⁸-Lys³³-Val³⁴-GLP-1(7-37)OH 0.08 Val⁸-Lys³³-Asn³⁴-GLP-1(7-37)OH 0.09Val⁸-Gly³⁴-Lys³⁵-GLP-1(7-37)OH 0.34 Val⁸-Gly³⁶-Pro³⁷-GLP-1(7-37)NH₂ 0.53

TABLE 9 GLP-1 receptor activation relative to PolypeptideVal⁸-GLP-1(7-37)OH GLP-1(7-37)OH 2.1 Val⁸-GLP-1(7-37)OH 1.0Gly⁸-GLP-1(7-37)OH 1.7 Val⁸-Tyr¹²-GLP-1(7-37)OH 1.7Val⁸-Tyr¹²-GLP-1(7-36)NH₂ 1.1 Val⁸-Trp¹²-GLP-1(7-37)OH 1.1Val⁸-Leu¹⁶-GLP-1(7-37)OH 1.1 Val⁸-Val¹⁶-GLP-1(7-37)OH 1.1Val⁸-Tyr¹⁶-GLP-1(7-37)OH 2.5 Gly⁸-Glu²²-GLP-1(7-37)OH 2.2Val⁸-Leu²⁵-GLP-1(7-37)OH 0.5 Val⁸-Tyr¹²-Tyr¹⁶-GLP-1(7-37)OH 1.5Val⁸-Trp¹²-Glu²²-GLP-1(7-37)OH 1.7 Val⁸-Tyr¹²-GIu²²-GLP-1(7-37)OH 2.7Val⁸-Tyr¹⁶-Phe¹⁹-GLP-1(7-37)OH 2.8 Val⁸-Tyr¹⁶-Glu²²-GLP-1(7-37)OH 3.6,3.8 Val⁸-Trp¹⁶-Glu²²-GLP-1(7-37)OH 4.9, 4.6Val⁸-Leu¹⁶-Glu²²-GLP-1(7-37)OH 4.3 Val⁸-Ile¹⁶-Glu²²-GLP-1(7-37)OH 3.3Val⁸-Phe¹⁶-Glu²²-GLP-1(7-37)OH 2.3 Val⁸-Trp¹⁸-Glu²²-GLP-1(7-37)OH 3.2,6.6 Val⁸-Tyr¹⁸-Glu²²-GLP-1(7-37)OH 5.1, 5.9Val⁸-Phe¹⁸-Glu²²-GLP-1(7-37)OH 2.0 Val⁸-Ile¹⁸-Glu²²-GLP-1(7-37)OH 4.0Val⁸-Lys¹⁸-Glu²²-GLP-1(7-37)OH 2.5 Val⁸-Trp¹⁹-Glu²²-GLP-1(7-37)OH 3.2Val⁸-Phe¹⁹-Glu²²-GLP-1(7-37)OH 1.5 Val⁸-Phe²⁰-Glu²²-GLP-1(7-37)OH 2.7Val⁸-Glu²²-Leu²⁵-GLP-1(7-37)OH 2.8 Val⁸-Glu²²-Ile²⁵-GLP-1(7-37)OH 3.1Val⁸-Glu²²-Val²⁵-GLP-1(7-37)OH 4.7, 2.9 Val⁸-Glu²²-Ile²⁷-GLP-1(7-37)OH2.0 Val⁸-Glu²²-Ala²⁷-GLP-1(7-37)OH 2.2 Val⁸-Glu²²-Ile³³-GLP-1(7-37)OH4.7, 3.8, 3.4 Val⁸-Asp⁹-Ile¹¹-Tyr¹⁶-Glu²²-GLP-1(7-37)OH 4.3Val⁸-Tyr¹⁶-Trp¹⁹-Glu²²-GLP-1(7-37)OH 3.5Val⁸-Trp¹⁶-Glu²²-Val²⁵-Ile³³-GLP-1(7-37)OH 5.0Val⁸-Trp¹⁶-Glu²²-Ile³³-GLP-1(7-37)OH 4.1Val⁸-Glu²²-Val²⁵-Ile³³-GLP-1(7-37)OH 4.9, 5.8, 6.7Val⁸-Trp¹⁶-Glu²²-Val²⁵-GLP-1(7-37)OH 4.4 Val⁸-Cys¹⁶-Lys²⁶-GLP-1(7-37)OH4.2 Val⁸-Cys¹⁶-Lys²⁶-Arg³⁴-GLP-1(7-37)OH 2.4, 1.9

EXAMPLE 10

In Vivo Comparison Dog Studies:

Sample 1: A 30:70 Val⁸-GLP-1:NPH mixture was prepared as described inexample 4. The mixture contained final concentrations of 0.48 mg/mlVal⁸-GLP-1, 2.45 mg/ml insulin-NPH, 1.6 mg/mL m-cresol, 0.65 mg/mLphenol, 10 mM phosphate buffer, and 16 mg/mL glycerin. The pH wasadjusted to a final pH of 7.8.

Sample 2: A U70 suspension of commercial insulin-NPH was prepared bydiluting U100 commercial insulin-NPH 30% with 1×NPH diluent. The U70suspension of commercial insulin-NPH contained final concentrations of2.45 mg/ml insulin-NPH, 1.6 mg/mL m-cresol, 0.65 mg/mL phenol, 14 mMphosphate buffer, and 16 mg/mL glycerin. The pH was 7.4.

Sample 3: A solution of Val⁸-GLP-1 was prepared as described in example4, but instead of mixing with commercial insulin-NPH suspension, theVal⁸-GLP-1 solution was mixed with: 1×NPH diluent at a ratio of 30:70(volume:volume) Val⁸-GLP-1:diluent. The 30% Val⁸-GLP-1 solutioncontained final concentrations of 0.48 mg/ml Val⁸-GLP-1, 1.6 mg/mLm-cresol, 0.65 mg/mL phenol, 10 mM phosphate buffer, and 16 mg/mLglycerin. The pH was adjusted to a final pH of 7.8.

A three arm pilot study was performed in dogs comparing the abovesamples. In the first arm of the pilot study, sample 1 was injected intoa single site on the neck of four different dogs at a dose of 0.74 U/kgNPH (1.5 nmol/kg Val⁸-GLP-1). A 3-hour hyperglycemic (150 mg/dl) clampwas initiated and glucose infusion rates were continually recorded.Blood samples were taken periodically for the determination of plasmaglucose, insulin, C-peptide, and immunoreactive GLP-1 concentrations.Plasma glucose concentrations were determined on the day of study. Theremainder of the samples were then frozen (−80° C.) and assayed forhormone concentration determinations at a later time.

In the second arm of the pilot study, sample 2 was injected into onesite on the neck of the same four dogs at a dose of 0.74 U/kg NPH. Alsoat the same time, sample 3 was injected into a second site on the neckof the same four dogs at a dose of 1.5 mmol/kg Val⁸-GLP-1. Again, a3-hour hyperglycemic (150 mg/dl) clamp was initiated and glucoseinfusion rates were continually recorded. Blood samples were takenperiodically for the determination of plasma glucose, insulin,C-peptide, and immunoreactive GLP-1 concentrations. Plasma glucoseconcentrations were determined on the day of study. The remainder of thesamples were then frozen (−80° C.) and assayed for hormone concentrationdeterminations at a later time.

In the third arm of the pilot study, sample 2 only was injected into asingle site on the neck of the same four dogs at a dose of 0.74 U/kgNPH. Again, a 3-hour hyperglycemic (150 mg/dl) clamp was initiated andglucose infusion rates were continually recorded. Blood samples weretaken periodically for the determination of plasma glucose, insulin,C-peptide, and immunoreactive GLP-1 concentrations. Plasma glucoseconcentrations were determined on the day of study. The remainder of thesamples were then frozen (−80° C.) and assayed for hormone concentrationdeterminations at a later time.

The results of the in vivo comparison dog study, shown in FIGS. 1-4,indicate that when Val⁸-GLP-1 was injected, there was a clear increasein glucodynamic activity over that observed when only insulin-NPH wasinjected (see FIG. 1). Surprisingly, the increase in glucodynamicactivity tended to be more robust and consistent with the first arm ofthe study as compared to the second arm of the study (see FIGS. 1 and2). However, the C-peptide and immunoreactive GLP-1 concentrations ofthe first arm were comparable to the second arm (see FIGS. 3 and 4,respectively). Also observed was a prolongation of absorption ofVal⁸-GLP-1 in the first arm of the study as compared to the absorptionof Val⁸-GLP-1 in the second arm of the study (see FIG. 4).

EXAMPLE 11

Exendin-4/Lantus® Mixtures:

Exendin-4 (160 μg) was dissolved in 100 μL of commercial Lantus® insulin(U00, 3.6378 mg/mL). The mixture was hand swirled gently until thesolids were dissolved and a clear solution was obtained. The solutionwas stored at 5° C. After 18 hours the solution was still clear withoutprecipitation. The pH was 4.0 Final concentrations of Exendin-4 andLantus® were determined by reverse phase HPLC to be 0.9 mg/mL Exendin-4and 3.5 mg/mL Lantus®. Based on known concentrations of excipients inthe commercial formulation of Lantus®, the final concentrations ofexcipients in the mixture were as follows: 2.7 mg/mL m-cresol, 30 μg/mLzinc, and 17 mg/ml glycerol.

EXAMPLE 12

Exendin-4/Lantus® Mixtures:

Exendin-4 (290 μg) was dissolved in 100 μL of commercial Lantus® insulinU100, 3.6378 mg/mL). The mixture was hand swirled gently until thesolids were dissolved and a clear solution was obtained. The pH of thesolution was adjusted to 4.0 with NaOH. Final concentrations ofExendin-4 and Lantus® were determined by reverse phase HPLC to be 1.6mg/mL Exendin-4 and 3.5 mg/mL Lantus®. Based on known concentrations ofexcipients in the commercial formulation of Lantus®, the finalconcentrations of excipients in the mixture were as follows: 2.7 mg/mLm-cresol, 30 μg/ml zinc, and 17 mg/ml glycerol.

EXAMPLE 13

Exendin-4/Lantus® Mixtures:

Exendin-4 (529 μg) was dissolved in 500 μL of commercial Lantus® insulin(U100, 3.6378 mg/mL). The mixture was hand swirled gently until thesolids were dissolved and a clear solution was obtained. The pH of thesolution was adjusted to 4.0 with NaOH. Final concentrations ofExendin-4 and Lantus® were calculated to be 1 mg/mL Exendin-4 and 3.5mg/mL Lantus®. Based on known concentrations of excipients in thecommercial formulation of Lantus®, the final concentrations ofexcipients in the mixture were as follows: 2.7 mg/mL m-cresol, 30 μg/mlzinc, and 17 mg/ml glycerol.

EXAMPLE 14

Val⁸-Glu²²-GLP-1(7-37)OH/Lantus® Mixtures:

A stock solution of Val⁸-Glu²²-GLP-1(7-37)OH was prepared by dissolving2.1 mg of Val⁸-Glu²²-GLP-1(7-37)OH in 1 ml of 0.01 N HCl. The pH wasadjusted to 11.3 with NaOH to obtain a clear solution. The pH was thenadjusted to 4.0 with HCl and remained clear. Val⁸Glu²²-GLP-1(7-37)OH(300 μL of stock solution) was added to 700 μL of commercial Lantus®insulin (U1100, 3.6378 mg/mL). The pH was adjusted to 3.3 with HCl toobtain a clear solution, and then adjusted to 4.2 with NaOH and thesolution remained clear. Final concentrations ofVal⁸-Glu²²-GLP-1(7-37)OH and Lantus® were determined by reverse phaseHPLC to be 0.5 mg/mL Val⁸-Glu²²-GLP-1(7-37)OH and 2.4 mg/mL Lantus®.Based on known concentrations of excipients in the commercialformulation of Lantus®, the final concentrations of excipients in themixture were as follows: 1.9 mg/mL m-cresol, 21 μg/ml zinc, and 11.9mg/ml glycerol.

EXAMPLE 15

Val⁸-Glu²²-GLP-1(7-37)QH/Lantus® Mixtures:

Stock solution A was prepared by dissolving 2.0 g of synthetic glycerin,0.3 g meta-cresol, and 120 μL of a 25 mg/mL Zinc oxide solution in 100mL of sterile water. The solution was filtered with MilliporeSterivex-GV 0.22 μm filter.

A stock solution of Val⁸-Glu²²-GLP-1(7-37)OH was prepared by dissolving4.8 mg of Val⁸-Glu²²-GLP-1(7-37)OH in 1.5 ml of. Stock solution A. ThepH was adjusted to 11 with NaOH. The pH was then adjusted to 4.0 withHCl and the solution remained clear. Val⁸-Glu²²-GLP-1(7-37)OH (300 μL ofstock solution) was added to 700 μL of commercial Lantus® insulin (U100,3.6378 mg/mL). The pH was adjusted to 2.7 with HCl to obtain a clearsolution, and then adjusted to 3.5 with NaOH. Final concentrations ofVal⁸-Glu²²-GLP-1(7-37)OH and Lantus® were calculated to be 0.96 mg/mLVal⁸-Glu²²-GLP-1(7-37)OH and 2.5 mg/mL Lantus®. Based on knownconcentrations of excipients in the commercial formulation of Lantus®,the final concentrations of excipients in the mixture were as follows:1.9 mg/mL m-cresol, 21 μg/ml zinc, and 11.9 mg/ml glycerol.

EXAMPLE 16

Exendin-4/A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg) Mixtures:

Stock solution A was prepared by dissolving 2.0 g of synthetic glycerin,0.3 g meta-cresol, and 120 μL of a 25 mg/mL Zinc oxide solution in 100mL of sterile water.

The solution was filtered with Millipore Sterivex-GV 0.22 μm filter.

A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg) (human insulin with Arg at position 0of the A chain, Gly at position 21 of the A chain, Arg at position 0 ofthe B chain, and Arg attached to the epsilon amino group of the Lys atposition 29 of the B chain) (384 μg) was dissolved in 100 μL of Stocksolution A. The pH of the solution was adjusted to 4.0 with NaOH.

Exendin-4 (197 μg) was dissolved in theA0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg) solution. The pH of the solution wasadjusted to 4.1 with NaOH. Final concentrations of Exendin-4 andA0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg) were determined by HPLC to be 1.1mg/mL Exendin-4, and 2.4 mg/mL A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg).

EXAMPLE 17

Exendin-4/A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg) Mixtures:

Stock solution A was prepared by dissolving 2.0 g of synthetic glycerin,0.3 g meta-cresol, and 120 μL of a 25 mg/mL Zinc oxide solution in 100mL of sterile water. The solution was filtered with MilliporeSterivex-GV 0.22 μm filter.

A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg)(human insulin with Arg at position 0of the A chain, Gly at position 21 of the A chain, Arg at position 0 ofthe B chain, and Arg attached to the epsilon amino group of the Lys atposition 29 of the B chain) (1.84 mg) was dissolved in 500 μL of Stocksolution A. The pH of the solution was adjusted to 4.1 with NaOH.

Exendin-4 (568 μg) was dissolved in theA0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg) solution. The pH of the solution wasadjusted to 4.2 with NaOH. Final concentrations are calculated to be 1.1mg/mL Exendin-4, and 3.68 mg/mL A0^(Arg)-B0^(Arg)-B29^(Lys-Nε-Arg).

1. A pre-mixed formulation comprising a GLP-1 compound and an acylatedbasal insulin wherein the GLP-1 compound isArg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37) and the acylatedbasal insulin is NεB29-tetradecanoyl des(B30) human insulin.
 2. Aprocess of preparing the pre-mixed formulation of claim 1, wherein theprocess comprises the steps of mixing said GLP-1 compound with saidbasal insulin in an aqueous medium.
 3. A method of treating a conditionselected from the group consisting of non-insulin dependent diabetes andinsulin dependent diabetes comprising administering an effective amountof a pre-mixed formulation of claim 1 to a patient in need thereof.